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KT

JOURNAL OF GENETICS

CAMBRIDGE UNIVERSITY PRESS

C. F. CLAY, Manageu

ILonDon: FETTER LANE, E.G.

eiinbtirgij: 100 PEINCES STBEET

aontlou: 11. K. LEWIS, i:ic, GOWKR STREET, W.C.

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All rights mserved

JOURNAL OF GENETICS

EDITED BY

W. BATESON, M.A., F.R.S.

DIRECTOR OF THE JOHN INNES HORTICULTURAL INSTITUTION
AND

R. C. PUNNETT, MA., F.R.S.

ARTHUR BALFOUR PROFESSOR OF GENETICS IN THE UNIVERSITY OF CAMBRinGE

Volume IV. 1914-1915

LIBRARY

^E^v YORK
«<>ta,\ical

tiAKUbA.

Cambridge :

at the University Press

1915

AT

I'KINTKIi HY JOHN CL.W, M.A.
AT THE UMVEKSITV PRESS

CONTENTS.

No. 1 (June, 1914)

PAOR

L. DoNCASTEH. On the Relations between Chromosomes, Sex-limited
Transmission and Sex-determination in Abraxas yrossulariata.
(With Plates I— III) 1

R. C. PuNNETT and P. G. Bailey. (Jn Inheritance of Weight in

Poultry. (With Plate IV and 3 Text-Figures) ... 2.3

H. M. Leake. A Preliminary Note on the Factors controlling the

Ginning Percent of Indian Cottons . . . . . . 41

N. I. Vavilov. Immunity to Fungous Diseases as a Physiological

Test in Genetics and Systeraatics, exemplitied in Cereals . 49

H. E. Jordan. Hereditary Lefthandedne.ss, with a Note on Twinning.

(Study III.) (With 80 Figures) GT

George Harrison Shull. A Peculiar Negative Correlation in

Oenothera Hybrids. (Witli Plates V and Vl and 1 Text-Figure) 83

M. Wheldale and H. Ll. Bassett. On a Supposed Synthesis of

Anthocyanin . . . . . . . . . .103

No. 2 (October, 1914)

M. Wheldale. Our present Knowledge of the Chemistry of the
Mendelian Factors for Flower-Colour. (With Plate VII, and
11 Text-Figures) 109

Clifford Dobell. A Comnientary on the Genetics of the Ciliate

Protozoa. (With 5 Text-Figures) 131

Arthur Ernest Everest. A Note on Wiieldale and Bassett's Paper
" On a Supposed Synthesis of Anthocyanin." (With 1 Text-
Figure) 191

vi ContentH

Nn. ?, (Januaiy, 1915)

PAGK

Frank C. Miles. A Genetic and Cytological Study of Certain
Types of Albinism in Maize. (With Plate VIII, and 9 Text-
Figures) 19;^

H. M. Fucil.s. Studias in the Plivsiology "f Fertilization . . 215

No. 4 (April, 1915)

R. P. Gregory. Note on the Inheritance of Heterostylisni in

Primula acaidis Jacq. ........ 303

R. P. Gregory. On Variegation in Primn.Ut sinensis. (With

Plates IX and X) 305

H. Dkinkwater. a Second Bracliydactylous Family. (With Plates

XI— XV, and 3 Text-Figures) 323

C. J. Bond. On the Priniar}' and Secondary Sex Characters of
some Abnormal Begonia Flowers and on the Evolution of the
Monoecious Condition in Plants. (With Plates XVI and XVII) 34 1

R. RuGGLEs Gates. On the Origin and Behaviour of Oenothera

ruhricalyx . . . . . . . . . .353

Arthur Ernest Everest. Recent Chemical Investigations of the
Anthocyan Pigments and their bearing upon the Production of
these Pigments in Plants . . . . . . . .361

M. Wheldale. Our Present Knowledge of the Chemistry of the

Mendelian Factors for Flower-Colour ..... 369

ERRATA.

Page 92, line ">,y'o/' "obvate" r"ad "ovate."

Page 104, line 4, for " + sugar x (rtavono)" read " + sugar .)• (flavone)."

Page 105, line 9, /')/• "witli roiiioval of sugar.s" read "vVilhout removal of .sugars."

Vol. 4, No. 1

June, 1914

JOURNAL OF GENETICS

EDITED BY

W. BATESON, M.A., F.R.S. *

DIRECTOR OF THE JOHN INNES HORTICULTURAL INSTITUTION
AND

R. C. PUNNETT, M.A., F.R.S.

ARTHUR BALFOUR PROFESSOR OF GENETICS IN THE UNIVERSITY OF CAMBRIDGE

CAMBRIDGE UNIVERSITY PRESS

C. F. CLAY, Manager

LONDON : FETTER LANE, E.G.

EDINBURGH: 100, PRINCES STREET

also

H. K. LEWIS, 136, GOWER STREET, LONDON, W.C.

WILLIAM WESLEY AND SON, 28, ESSEX STREET, LONDON, W.C.

PARIS : LIBRAIRIE HACHETTE & c"^.

BERLIN : A. ASHER & CO. LEIPZIG : BROCKHAUS

CHICAGO : THE UNIVERSITY OF CHICAGO PRESS

BOMBAY AND CALCUTTA : MACMILLAN & CO., LTD.

TORONTO : J. M. DENT & SONS, LTD.

TOKYO : THE MARUZEN-KABUSHIKI-KAISHA

Price Ten Shillings net

Issued July 7, 1914]

The University of Chicago Press

Artificial Parthenogenesis and Fertilization. By Jacques
LoEB, Member of the Rockefeller Institute for Medical Research.
318 pages, 12mo, cloth; 10s. net.

This new work presents the first complete treatment of the subject of artificial
parthenogenesis in English. Professor Loeb published foui- years ago a book in
German under the title Die chemische Entioickiungierregung des tierischen Eies.
Mr W. 0. R. King, of the University of Leeds, translated the book into English,
and the translation has been revised, enlarged, and brought u]3 to date by Professor
Loeb. It gives, as the author says in the preface, an account of the various methods
by which unfertilized eggs can be caused to develop by physico-chemical means,
and the conclusions which can be drawn from them concerning the mechanism by
which the spermatozoon induces development. Since the problem of fertilization is
intimately connected with .so many different problems of physiology and pathology,
the bearing of the facts recorded and discussed in the book goes beyond the special
problem indicated by the title.

British Medical Journal. The subject of the book is an analysis of the mechanism by
which the male sex cell — the spennatozoon — causes the animal egg to develop. The
author has gained a world-wide reputation for his achievements in artificial fertilization,
and this work shows how, according to his observations, the action of well-known chemical
and physical agencies may be substituted for that of the living spermatozoon.

Heredity and Eugenics. By John M. Coulter, William E.
Castle, Edward M. East, William L. Tower, and Charles B.
Davenport. 312 pages, 8vo, cloth ; 10s. net.

Five leading investigators, representing the University of Chicago, Harvard
University, and the Carnegie Institution of Washington, have contributed to this
work, in which great care has been taken by each contributor to make clear to the
general reader the present position of evolution, experimental results -in heredity in
connection with both plants and animals, the enormous value of the practical
application of these laws in breeding, and human eugenics. Technicalities of
language have been avoided, and the result is an instructive and illuminating
presentation of the subject for readers untrained in biology as well as for students.

Contents : I. Recent Developments in Heredity and Evolution : General Introduction.

II. The Physical Basis of Heredity and Evolution from the Cytological Standpoint (John
Merle Coulter, Professor and Head of the Department of Botany, the University of Chicago).

III. The Method of Evolution. IV. Heredity and Sex (William Ernest Castle, Professor
of Zoology, H.irvard University). V. Inheritance in Higher Plants. VI. The Application
of Biological Principles to Plant Breeding (Edward Murray East, Assistant Professor of
Experimental Plant Morphology, Harvard University). VII. Recent Advances and the
Present State of Knowledge concerning the Modification of the Germinal Constitution of
Organisms by Experimental Processes (William Lawrence Tower, Associate Professor of
Zoology, the University of Chicago). VIII. The Inheritance of Physical and Mental
Traits of Man and their Appheation to Eugenics. IX. The Geography of Man in
Relation to Eugenics (Charles Benedict Davenport, Station for Experimental Evolution,
Carnegie Institution of Washington).

British Medical Journal. Those who are desirous of arriving at an estimate of the
present state of knowledge in all that concerns the science of genetics, the nature of
the experimental work now being done in its various departments, ...and the prospects,
immediate or remote, of important practical applications, cannot do better than study
"Heredity and Eugenics."

The Nation, New York. "Heredity and Eugenics" may be heartily recommended to
readers seeking, as beginners, to get in touch with the discussion of these subjects. ... In
most of the lectures there is an admirable reserve, not to say skepticism, in the treatment
of large questions which the public is often misled to regard as already and finally settled.

The Cambridge University Press

Agents for the British Empire

London, Fetter Lane

Volume IV JUNE, 1914 No. 1

ON THE RELATIONS BETWEEN CHROMOSOMES,
SEX-LIMITED TRANSMISSION AND SEX-
DETERMINATION IN ABRAXAS GROSSU-
LARIATA.

By L. DONCASTER, Sc.D.,
Fellow of King's College, Cambridge.

In the last of a series of papers on chromosomes and sex in
Abraxas grossulariata (Currant Moth), I described breeding experi-
ments with a strain which in each generation produced families
consisting entirely of females, and showed that in this strain all
the ovaries examined, with one exception, had oogcmia with 55 chro-
mosomes instead of 56, the normal number in the species'. The
present paper confirms and amplifies the results previously described,
gives an account of the chi-omosomes in the maturation of the egg
in normal females and in the strain which produces unisexual broods,
and describes an exception to the normal sex-limited transmission of
the grossulariata character which can possibly be correlated with an
exception in the chromosome number in such a way as to suggest
a definite relation between that character and a chromosome. It
also gives direct evidence of dimorphism of the eggs in respect of
chromosome number, exactly comparable with the dimorphism of sper-
matozoa which has now been described in so many insects of other
orders.

Material and Methods.

The methods adopted were in general the same as described in
my previous papers, except that the larvae (usually about half-grown)
were dissected in most cases in tap-water instead of Ringer'.s fluid.
I found by accident that this gave clearer division-figures ; I discovered
that the Ringer's fluid used for the ovaries obtained in the autumn

' Journal of Genetics, VoL in. 1913, p. 1.
Journ. of Gen. iv 1

2 Chromosomes and Sex hi Abraxiis

of 1912 had accidentally been made up by my assistant to only one-
tenth of the normal strength, and on making up the correct solution
it gave very inferior results. Tap-water gave the best results of all.
Sections for both ovarian and maturation divisions were stained with
Heidenhain's Iron Haematoxylin ; in the latter case the stain nmst
be washed out until the yolk is left almost colourless.

For the maturation of the eggs, I have to thank Miss P. H. Dederer
of New York for giving me full information in writing about the
methods used by her in studying the maturation divisions of Philo-
samia cynthia. I followed her methods in general, with modifications
due to the much smaller eggs and thinner shells of Abraxas grossu-
lariata. The eggs were fixed at varying times up to about 3 hours
in Carnoy's alcohol sublimate (absolute alcohol, glacial acetic acid,
chloroform, in equal parts ; sublimate to saturation). They were then
washed in 70 % alcohol, and usually treated with iodine in 70 or 90 %
alcohol to remove the sublimate, and preserved in 80 J^ alcohol. For
cutting sections it is necessary to remove the shell, always a trouble-
some and often a difficult operation. In some batches the egg is
contracted away from the shell, and the shell can then be removed
without much difficulty with needles under a binocular dissecting
microscope. In some cases, eggs which had been treated with iodine
seemed to have softer shells, but I am not sure that this is general.
The removal of the shell seemed usually to be considerably e;isier in
eggs which had been kept for some weeks in spirit. In other liatches
the protoplasmic layer of the egg sticks to the shell, and to remove
it without tearing it from the yolk is a matter of gi-eat difficulty. If it
is once separated from the yolk, accurate orientation becomes impossible
and the polar region often cannot be found in the .sections. The polar
spindles are at the anterior end of the egg, w'hich in this species can
generally be recognised by the less marked sculpturing of the shell.
The shell was pricked and torn apart from the posterior end when this
could be identified. The shelled eggs were passed through absolute
alcohol into cedar oil for some hours or more, placed in xylol for a few
minutes, and embedded in paraffin melting at 60'. The sections were
cut transversely as a rule, with a thickness of 10 /i.
a. Breeding Eivperiinents.

The main facts established in the previous paper were that in a
certain strain families appeared in each generation consisting entirely
of females ; some of these females again have only female offspring,
while others, apparently of about equal number, produce males and

1907

1908

ngs
ake

1909

1910

1911

1912

'10.15 3.IO
Li X G ,x Li

32 J, 27 14?

The <Iescendant9 of '10.10 include

'11.11 (lOrf, '20?)

'11.6 ( 7d-, 16?)

'11.13 ( 9j, '21 ? )

'12.10 (.35 rf, 46?)

'12.16 (32 <f , 43 ? )

'12.20 (33 J, 42? )

'12.42 (36 J, 47?)

'ILO-ll '11.3 '11.12 '11.24 ^'11.24A '11.24 B

G ? X I xG i L?xG(f\vild Li X Li L <t x L s Li x Li L 1 x Li

l.'y-i i , 7 ? 19 cf , 15 ?

'12.32 '12

12.29 12-29 e

} ? X wild i G 1 X wild s

6fi?

4 rf , 5 ? 23 cf , 16 ? 27 J , 27 ? '24 j , 21 ?

'12.27 B

'12.36 '12.33 '12.37

L ? X wild i L 1 X L i

iOi , 5 ? 88 J , ofi ?

'10.16 UO.lli

/. SxOrf rjxOrf wild

■f , 27 «

■i2 rf , 19 !

•11

9

■ll.SS

'11.14

0 i X
IS

>

^ 8 y Grf
9»

ISi, 33 «

•la.S'i '12.11 '12.1M 'IS.SI '12.15

TABLE I.

Pedit/refi of All-Jifmale .Strain.

(Alt-female brooda in italicK, broode with great excess of females in thick t>'i)e.)

Only the ancestry in the female line is shown ; the ancestry of the male parents can be found in the Tables of Matinps
in thiH and the preceding paper. The years are those iu which the matings were made. G = gross., /. = lact. For the sake
of clearness, the number of offspring in the normal families of the 1912 matings arc not given ; they will be found in
the Table of Matings.

'07.19

Wild Gi x La

•10.11

•10.H3

•10.24

■10.25

•10.27

•lO/JH

■10.17

'lO.lO

Li X Li
Hi

Li xOj
4t

Li xLi

A

22 i. 26 ?

Li X Oj

A

19 tT , 23 s

Li xOi

A,

Li X r,i

62 1

Li X Li

A

4 ^ . 60 ;

Li ^ Li

.A

Tho rloBcomlnnla ot •lO.lll iiKlii
'11.11 (lOj, '20?)
'11,6 ( 7<r, 16!)
•11.13 ( 9<(. 21 ? I
•12,10 (35 .f, 16!)
'12.16 (32 / . 13 ! )
'12.20 (33 i , 42 ! )
'12.42 (36.;, 47!)

■il.4 'ILlS

fl 1 X Li Gl X Gi

'H.IB '11.21 'il,J6

Li X Li O 1 X Li Li X n i \vi)il

A A 1

li , •/ 1 a , 11

'11.28
01 xOi

11.30 '11.1 '11.3 '11.12 '11.24 'll.'.ilA '11, '.'IB

1 X Li Li X G i Li X G i wild LI X Li Li x Li Li x Li hi x Li

A A A A

15 » 4 rf , 5 ! 23 ^ , 16 ! 27 i , 27 9 -li .! , 21 !

AAA/

UtT, 17! 23 <f, 201 12 i, 71 19 i

'12.'26 '12.40 'la.l '12.3 '12.0 '12.9 B 'JJ.S5 '12.25 •12.8 •i2.l9 '12.21 'l2 2lB

LI xGi Li X Li Li xGi Li x Gi Ol xGi Gl x Gi wild Gl x Li G 1 , L i

li. 16! 10 J, 18!

A A

Xi. Oe t 32 J, 14!

•11.29 12-29B

G 1 X wild j 0 1 X wild i

'12.36 'T2.3H •rj.37 '12.27 B

L 1 X wild i Li xLi

lai . 82 7 2i 25 <

J , 56 !

L. DONCASTER

TABLE II. Mathigs, 1912-1913.

This table is a continuation of that given for previous years, Journ. of Genetics, Vol. ni. 1913,
pp. 4, 5. Reference numbers of purely female families are given in italics ; those of families with at
least twice as many females as males in thick type. Since a number of larvae were dissected for
cytological examination, the figures in the 'Total Males' and 'Total Females' columns are often
larger than the sum of 'gross, males ' + 'lact. males,' etc.

Iinagos

Reference
Number

Female Parent

Male Parent

Number
of EgKs

(iross.
Males

Lact.
Males

Gross.
Females

Lact.
Females

Total
Males

Total
Females

'12.8
'12.8 (2)
'12.19
'12.29
'12.29 B

gross, ex '11.26

gross, ex '11.11
wild

65

42

98

111

about 83

—

—

7

4

38

34

52

9

7

—

36

28
57
77
66

'12.35

lact. ex '11.1

gross, ex '11.30

not counted

—

—

4

2

—

6

'12.1

i> )»

lact. ex '11.6

97

—

—

—

41

1

66

'1215
'12.21

gross, ex '11.14
gross, ex '11.26

gross, ex '11.30
lact. ex '11.24 A

58
73

6
2

—

8

7
25

7
4

16

37

'12.21(2) „
'12.21 B

t» )'

about 94
53

7

—

—

37
21

9
2

45
23

'12..3

lact. ex '11.1

lact. ex '11.11

about 100

—

16

—

28

33

28

'12.5

lact. ex '11.6

gross, ex '11.28

„ 80

17

—

18

—

26

26

'1'2.6

,, ,,

lact. ex '11.12

„ 100

—

35

—

24

40

29

'12.7

' J n

gross, ex '11.14

76

21

23

28

13

47

46

'12.9
'12.9(2)

lact. ex '11.1

gross, ex '11.3

38
41

7
4

11
6

5
3

3
2

21
11

9
5

'12.9 B

" ,,

,, ,,

about 30

7

4

3

5

12

8

'12.16

lact. ex '11.6

gross, ex '11.14

„ 50

15

3

12

18

19

30'

'12.16 (2)

'J >)

i» »i

„ 36

6

4

4

8

13

131

'12.18

lact. ex '11.2S

gross, ex '11.28

„ 85

17

—

13

—

30

14

'12.20

gross, ex '11.11

lact. ex '11.14

121

33

—

—

39

33

42

'12.10

,, ,,

11 11

about 90

22

—

—

22

26

40

'12.22
'12.22 (2)

gross, ex '11.13

lact. ex ll.'22A

39
65

12
3

—

—

15

7

17
13

17
13

'12.42

11 >i

lact. ex '11.24

109

33

_-

—

41

36

47

'12.25

gross, ex '11.15

lact. ex '11.14

69

21

—

2

14

31

23 2

'12.26

'12.26(2)

'1'2.27B

lact. ex '11.14
gross, ex '11.3

gross, ex '11.13

)t It
lact. ex '11.24 B

36
33
65

19

2
1

1

7

4

17

9
1

20

12 3
63
17

'12.31

lact. ex '11.25

wild

68

12

—

7

—

17

13

'12.32

lact. ex '11.9

,,

40

11

—

3

—

13

10

'12.32(2)

>t »)

11 »»

45

6

—

3

—

6

6

'12.40

lact. ex '11.14

78

19

1

14

1

25

22^

'12.41

lact. ex '11.9

lact. ex '11.6

26

—

6

—

3

11

4

'12..H3

lact. ex '11.30

wild

about 45

20

—

5

—

20

5

'12.37

>> )»

lact. ex '11.24 B

not counted

82

—

49

—

88

56

'12.28

wild

lact. ex '11. '24 A

83

29

—

—

29

33

31

'12.14

)j

gross, ex '11.21

48

17

—

8

12

23

22 =

'12.30

1)

lact. ex '11.14

95

19

—

—

27

20

2T>

'12.39

,,

55 Jt

about 75

30

—

—

25

30

27 ^

1 The inverted ratio of gross. : lact. in males and females is noteworthy.

2 Note two exceptional gross, females.

3 Note great excess of lact. where equality is expected. Some larvae of '12.26 (2) were lost at
hatching.

* The male and female lact. are unexplained exceptions.

' The object of these experiments was to determine whether a male immediately descended from a
unisexual family might produce unisexual offspring by a wild female.

1—2

Chi'omoHome>i <ni(l Sex in Abraxas

o

females in the inirnial ratio. The tendency to produce only fenial
offspring has usually descended in the direct female line, ])ul has
occasionally missed a generation, so that there were one or two cases
of females of bisexual families having only female offspring, and others
where such females had families in which males were produced, hut
only in extremely small numhers.

The complete pedigree of the all-female strain is given in Table I.
The details of the 1912 matings are given in Table II ; those of previous
years have been given in the earlier paper. Table II includes the
results of all the matings made, with the exception of some which were
nearly or quite sterile, or which have no close bearing on the (piestions
discussed in the present paper. As in the previous paper, the reference
numbers of families consisting entirely of females are printed in italics,
in both the table and the text; families in which there were at least
twice as many females as males are distinguished by thick type.

The moths reared in the year 1912-13 (from pairings made in
1912) confirmed the previous results, but added some new f;icts.

The chief new point of importance is the fact that broods which are
not entirely of one sex, but in which the females greatly outnumber
the males, may arise among the offspring of females which belonged to
unisexual broods (e.g. '12.1, '12.21, '12.21 B). It appears, in fact, that
there is no sharp distinction between uTiisexual broods and broods in
which the sexes are in nearly equal nimibers, but that various grades
of excess of females occur, leading to the extreme condition in which
all the offspring are female. These results therefore suggest that the
existence of unisexual broods is not due to the absence of a male-
determining factor from the mother, but that there is a tendency, of
varying amount, for this f;xctor to be eliminated from the egg. The
facts of sex-limited inheritance have already led to the conclusion that
the female in Abraxas is heterozygous for a sex-factor, and in normal
families it must be supposed that this factor is present in half the eggs
and absent from the other half In the strain which produces female
families it may be supposed that the factor, whatever it may be, which
determines maleness, has a tendency to be eliminated, in some cases
from all the eggs, in others from much mori' than half, instead of its
retention or elimination being a matter of chance. It will be seen
below that this su{)position is to some extent supported by cytological
observations on the maturation of the eggs.

There are one or two further points in the 1912-18 families which
require notice before passing on to the cytological observations. In

L. DONCASTRR 5

two families ('12.25, 12.40) there were exceptions to the ordinary
sex-limited transmission. These will be referred to more fully below.
It will be noticed that in several families the same reference number
is used, followed either by ' (2) ' or by ' B.' Such families as '12.8 and
'12.8 {2) wera reared from eggs of the same parents, 'li2.8 being from
the first eggs laid, '13.8 {'3) from eggs laid later. The object of this
was to determine whether the earlier and later eggs gave similar
results as regards sex-ratio. The results show that there is never any
important difference between the ratios of the sexes from the two lots.
When a family is distinguished with 'B' (e.g. '12.21, '12.21B), it was
reared from parents which were brother and sister of those of the
family with the same number without ' B.' Thus the male parent
of ' 12.21 B was brother of the male parent of '12.21, the female
parent sister of that of '12.21. Such pairs of families are in each
case closely similar in the 1912-13 families, as they were in previous
years, but the fact that two sisters mated to males of closely similar
ancestry sometimes give very different progeny (e.g. '12.1 and '12.3)
makes it doubtful whether the resemblance between the offspring of
two sisters mated to two brothers will be found in every case.

There were no cases in 1912-13 of females belonging to bisexual
broods having only female offspring, such as occurred in three families
in the preceding year. Matings of females belonging to broods in
which males were very scarce usually gave a smaller preponderance
of females, when the sex-ratio differed from equality, than occurred
in the mother's family (e.g. '12.16, '12.20); there were no cases of a
great increase in the ratio of females to males. It seems, therefore,
that when the tendency to produce female offspring is not strong
enough to cause all the progeny to be female, it suffers a progressive
decrease in intensity in subsequent generations.

Complete or nearly complete sterility occurred in six matings with
females of unisexual families, out of a total of 21 such matings, and
in four out of 23 other cases. The infertility of females of unisexual
families is thus not much greater than that of related moths belonging
to bisexual broods.

It may be mentioned incidentally that among the larvae of family
'12.27 B, seven were almost totally black. The moths when hatched
showed no peculiarity. Two of them paired together gave a brood of
23, which were all nearly or quite black. On the other hand, a male
and a female derived fi-om black larvae, paired with moths derived from
normal larvae, both gave broods consisting only of normals. The black

G Chromosomes and Sex in Abraxas

character is therefore probably a recessive mutation, but shows no sign
of being sex-limited in transmission. The Rev. G. Waddington, S.J.,
writes to me that he also finds evidence that the black form is recessive
to the type. In a brood of black larvae, the completeness of the
blackening varies somewhat in different individuals, bat even those
with least black are conspicuously different liom the normal.

h. The Oor/omal Chromosomes.

In my former paper on the strain which produces uni.sexual families,
I gave evidence that females belonging to unisexual broods have 55
oogonial chromosomes instead of the 56 which I had previously shown
to be the usual number for the species. The evidence consisted chiefly
in the observations that of brood 'JJ.S, 33 larvae were dissected, all
of which were females ; nine of these larvae provided altogether 18
oogonial mitotic figures, in which the number 55 was almost or quite
certainly correct, and in other larvae a number of figures were found
in which 55 was the most probable number. In female larvae of broods
'12.1 and '12.32 also, five figures were found each with 55, and several
others in which 55 was most probable but not aksolutely certain. Since
of these broods '12.8 was the only ope which was known at all certainly
to consist only of females (many larvae being still too small to dissect)
it was concluded that all females belonging to unisexual broods had
55 instead of 56 chromosomes, but that it was doubtful whether females
belonging to bisexual families descended directly from unisexual iiad
always 55, or sometimes 55 and sometimes 56.

The work during the spring of 1913 has definitely shown that not
only females of unisexual broods, but also females of bisexual broods
which are inmiediately descended from unisexual broods, alwaj's have
55, with one probable exception to be referred to later. In the following
account the observations previously recorded on brood 'li^.8 will not
be described again, but the cases of broods '12.1, '12.25, and '12.32,
mentioned in the former paper, will be included together with the
observations made in 1913.

In all, in addition to the 33 female larvae of 'Ji'.S recorded in the
former paper, the ovaries of about 160 female larvae were dissected out
and sectioned. Of these 39 larvae belonging to the unisexual strain,
and eight not belonging to this strain, yielded ovaries with figures
considered good enough to count. Of the larvae belonging to the
unisexual strain, eleven belonged to three broods {'1:^.19, '1J.39,
'12.29 B) which consisted entirely of females; fifteen belonged to

L. DONCASTER

broods '12.1 and '12.21, in which females were in very great excess,
and the remaining thirteen to broods '12.18, '12.25, '12.31 and '12.32,
in which males and females were in approximately equal numbers.
In every one of these 39 larvae, with one exception which will be
discussed below (brood '12.25), all the really good figures showed 55
chromosomes (Figs, 3 — 8). The following is an analysis of the counts ;
all were made by drawing the chromosomes and then counting the
chromosomes drawn, and in many cases the figures were drawn again
later and the counts compared. I have classified the figures by notes
made at the time into five grades of trustworthiness — 'excellent,' 'good,'
' fair,' ' probable ' and ' doubtful ' — the ' doubtful ' group including those
in which it was not possible to decide with any confidence between 55
and 56. Classes 'excellent' and 'good' include cases where no reasonable
doubt is possible.

The counts in the unisexual broods were as follows. Individual
larvae are represented with letters (A, B, BB etc.) ; the number of
ovarian mitoses of each class is given after the letter in words.

Excellent
Total 8

Good
Total 10

Fair
Total 29

'13.39 iJJ, one.

'12.21 ? B, one.

'12.18 ■} D, one; f F, two; f G, one.

'12.31 iF, two.

'13.39 ? CC, one.

'12.1 ? fl, two; ? S, one; ? A', one.

'12.21 ? C, one; ? I, two.

'12.31 ? A, one.

'12.32 ¥J, one.

'13.39 ?B, two, ? f/, one; 1 BB, two;

'13.39 B iF, one.

? CC, one; ? II, one.
? K, one; ? N, one.

'12.21 ?B, two; ?F, three; ? G, three;

'12.1 i A, one; ? D, two; ? JF, one.

'12.18 i G, one.

'12.31 iA, one; ? £, two; ? F, two.

'12.32 ? B, one.

•13.39 ? O, one.

'13.19 ? B, one.

'12.21 ?</, three; ^ K, one.

'12.31 iD, one; i E, two; ? F, one.

'12.32 i A, one; ? C, one; i N, two.

'13.39 JL, one; ? GO, one.

'12.21 ? G, one; ? ^V, one.

'12.31 $ F, two.

'12.18 ?D, one.

In addition to the above there is one figure, in an ovary of a larva

($ /) of brood '12.1, in which 56 appears the most probable number.

Probable
Total 14

Doubtful
Total 7

8 Chromosomes and Sex in Abraxas

but the chromosomes have begun to divide, so that several appear
double, and it is not impossible that two which appear to be distinct
are really separating halves of one. Brood '12.25 is not included in
the list ; the exception which occurs in it is discussed below.

It appears from this list there are eighteen really good figures in
which the number 55 is perfectly clear, twenty-nine others in which
it is scarcely less certain, fourteen in which it is the most probable, and
only seven or eight in which it is doubtful ; and it should be noted that
of these doubtful figures, five are in ovaries of larvae in which the better
figures were counted as 55. I think, therefore, that on this evidence alone
it may be taken as certain that females of the unisexual strain, whether
they themselves belong to unisexual or bisexual broods, have 55 chro-
mosomes. Confirmatory observations from the maturation-figures of
the eggs will be described below.

Comparatively few larvae with mi descent in the direct female line
from unisexual families were examined, since the number 56 had been
determined with confidence as characteristic of the species in previous
work'. Of those from which ovaries were sectioned, two wild fjmales,
three females from brood '12.10, and one from brood '12.22 provided
figures which could be counted; all showed 50 (Fig. 1), and were dis-
tributed in the various grades as follows:

Excellent Wild ? B, one.

Total 3 ., ? C, one.

„ ? D, one.

Good '12.10 ? A, one.

Total 4 '12.22 ? C, three.

Fair '12.10 ? A', one.

Total 2 '12.22 ? C, one.

Probable '12.10 t O, two.
Total 2

These seven larvae, therefore, provided seven good or excellent
figures with 56, and four others in which 56 was almost certainly
the true number.

One very important point appt'ared in addition. One wild larva
(^ A) not included in the list just given, provided four ovarian mitotic
figures, three of them of the ' excellent' and one of the ' probable ' class,
all with 55 chromosomes (Fig. 2). Two of them had, in addition, one
or two minute stained specks on the edge of the chromosome group,
in such a position that they might be regai'ded as chromosomes if they

' Journal of Genetics, Vol. ii. 1912, p. 189.

L. DONCASTER 9

were not so much smaller than the normal chromosomes of the species.
Such specks are not infrequent on the edge of the spindle, and probably
have no connexion with chromosomes. In this female, therefore, it
must be concluded that only 5.5 normal chromosomes were present,
unless possibly a very much reduced 56th is represented by one of the
specks mentioned.

All these facts taken together lead to the conclusion that in Abraxas
gross id ariata the commonest chromosome number in the feuiale is 56,
but that in some strains one of these may be very much reduced or
absent. There is no necessary connexion between the number 55 and
the tendency to produce only female offspring, since all individuals
of the female-producing strain have 55, whether they belong to uni-
sexual or to bisexual broods. The nature of the mechanism which
results in unisexual families will be dealt with in connexion with the
account of the maturation divisions (below. Section d).

c. Spermatogenesis of the strain which has 55 oogoniul chromosomes
in the female.

In my first paper on the spermatogenesis of Abraxas grossulariata,
I described 28 chromosomes in both first and second spermatocyte
divisions ; I found no spermatogonial figures which could be counted
quite accurately. The testes then examined belonged to individuals
of the normal form with 56 chromosomes in both sexes, but it was
clearly a point of importance to find out whether the same number
occurs in males of the strain in which the female has 55.

I have examined a number of testes of broods '12.3, '12.18, '12.25,
'12.26, '12.31, all of which families are either known to have females
with 55, or are descended from the same stock. In all of the numerous
first sjjermatocyte figures counted there are clearly 28 chromosomes',
but this would probably be the case whether the spermatogonial
number were 56 or 55. The number in the secondary spermatocyte
divisions is somewhat less certain, but there is practically no doubt
that it is also 28 in all cases. In testes of the broods mentioned, be-
longing to the 55-chromosome strain, I have counted 41 good secondary
spermatocyte equatorial plates ; twenty-nine of these have quite clearly
28, and twelve have 26 distinct chromosomes and a pair of chromosomes
in contact. In males of two broods ('08.10, '12.7) not belonging to the
55-chromosome strain, the condition with two chromosomes in contact

' The only exception I have seen is in one figure in a testis of a male of '12.7, which
does not helong to the 55-chromosome strain. This single figure has 27, but many other
figures in the same testis have 28.

10 Chromosomes and Sex in Abraxas

seems to be somewhat more frequent ; I have counted ten figures with
28 separate chromosomes, and eight in which two are associated. The
degree of association varies somewhat, and in a few cases, especially in
one male of '12.7, the two are so close together that they might eiisily
be mistaken fur one. This is not the case in any of the figures from
the .55-chromosome strain, and since such a double chromosome is not
found in figures in which 2lS separate chromosomes ai-e seen, and since
the two members of the pair are not always equal in size, I think it is
certain that 2(S is the number constantly found in all seciiiidary sper-
matocytes. In Biston hirtmia I have found that in the si^eruiatDcytes
two chromosomes are constantly associated, and the occasional figures
which might be counted as 27 in Abraxas are probably due to the less
regular occurrence of the same sort of thing.

The spermatogonial divisions are not usually clear enough to give
quite reliable counts. I have nuxde very many attempts, and in almost
all the best figures I have found .56, whether of the strain with 55 in
the female or in males not belonging to this strain. I have altogether
twenty spermatogonial figures recorded as ' good ' or ' very good ' in
males belonging to the strain in which the female has 55 ; of these
sixteen show 56, three might be either 56 or 55, and one shows only 55.
All the six best figures have clearly 56 (Fig. 9). I have thereft)re no
hesitation in concluding that in all males 56 is the spermatogonial
number, and that all spermatozoa contain 28.

d. The Maturation Divisiovs of the Er/tjs.

The polar divisions take place in a little patch of protoplasm at
the anterior end of the egg, and in searching for them in sections the
anterior end can usually be recognised by the somewhat finer granu-
lation of the yolk. During the maturation, the superficial protoplasmic
layer of the egg is drawn out, just at the point where the outer polar
nucleus will lie, into a fringe which in sections has exactly the ap-
pearance of a tuft of cilia (Fig. 14) ; this usually makes the division-
figures easy to find in a longitudinal section, but is of no help in
sections transverse to the axis of the spintUes. I have not discovered
the meaning of the fringe ; it is possibly due simply to the superficial
protoplasm sticking to the shell at the point where the polar divisions
occur. During the maturation divisions, the heads of the spermatozoa,
with their radiations, are usually conspicuous objects in the jolk near
the anterior pole. One can almost always be found, and not rarely one
fir two others are present in addition. I cannot give the exact times

L. DONCASTBR 11

at which the various stages of the polar divisions take place, for the
usual method in fixing the eggs was to determine as accurately as
possible when the moth began to lay, and about two and a half hours
later to fix all the eggs laid. In eggs fixed less than an hour after
laying, usually the first polar spindle is found ; in eggs fixed two and a
half hours after laying began, the latest stage found is commonly the
anaphase of the second division. The equatorial plate stage of the second
division appears to last for some time, since it is the stage most fre-
(juently found. This is fortunate, for it is the only stage at which
the chromosomes in the inner and outer spindles can be counted with
certainty. Even then, on the average not more than one egg in ten
has the chromosome groups cut in a plane in which they can be
counted, so that of nearly 600 eggs cut and examined, only about
60 gave at all reliable counts. The proportion is considerably higher
in batches of eggs which could be shelled easily without damaging
them, so that accurate orientation is possible ; in such batches, as many
as one in three or four may give good iigures.

During the first polar division, a mass of granules which stain
deeplj' with iron haematoxylin is left in the equatorial plate as the
chromosomes travel to the poles (Fig. 14). This remains in the second
division as a flat plate of fine granules between the outer and inner
mitotic figures. When, as happens sometimes, one chromosome lags
behind the others, it can easily be determined to which spindle it
belongs, even when it is in the next section to the remainder of the
chromosomes, by its position on the outer or inner side of the granule
layer. In most cases, however, all the chromosomes of a group (outer
or inner equatorial plate) are found in one section, and except where
the contrary is stated, all the figures have been drawn from such cases.

One abnormality which was observed must be mentioned, since it
may have considerable theoretical importance. In one egg of brood
'13.4, and in two of brood '13.42, there are two separate polar division-
figures, near together at the anterior end of the egg (Figs. 15, 16 a, b).
In the egg of '13.4 and in one of those of '13.42 each figure is in the
equatorial plate stage of the second division, with an inner and outer
spindle; in the second egg of '13.42, each figure shows three vesicular
polar nuclei, and two female pronuclei each conjugating with a sperm-
nucleus (Fig. 16 «, b). There must therefore have been two nuclei in
these eggs, which are undergoing maturation simultaneously. If in such
an egg one nucleus underwent maturation in such a way as to give rise
to a male-producing pronucleus, the other to a female-producing, a

12 Chromosomes ami Sex in Abraxas

bilateral gynandromorph, such as is not unconimoii in Lepidoptera,
might be produced. Several suggestions have been made by various
writers as to the origin of such gynandromorphs, but a binucleate egg
of this kind seems as likely a cause of their production as any which
has been suggested. In this connexion, reference may be made to
the fact recorded in my previous paper on the chromosomes in Ahraxan
{Juuni. of Genet, ill. 1913, p. 9), that in several testes of family '12.2.5,
binucleate spermatocytes were not infrequent. Incidentally, Fig. 16 b
illustrates a point of considerable cytological interest. Around the
conjugating pronuclei there are conspicuous radiations in the y<)lk,
and it is clearly seen that these have a somewhat sinuous course in
the protoplasm between the alveoli which contain the yolk-granules.
The protoplasm near the pronuclei, and also around the polar nuclei,
strongly suggests that their radiations are caused by outgrowths of
protoplasmic alveoli, as suggested by Jenkinson'. The radiations are
not only sinuous, but in several cases are clearly branched. Further,
since the two pairs of pronuclei are near together, their radiations are
intermingled, and in some cases those belonging to one system quite
clearly cross those of the other. Such crossing can be seen in one section,
but I have found no case of two radiations crossing when both are
in focus ; since, howevoi-, the section is 10 ^ thick, and the crossing
radiations are well within the thickness, they cannot be much more
than 5 fi apart. As far as can be seen, the two sets of radiations
spread from centres quite independently of each other, like the roots
of two plants growing near together.

I have examined eggs of only one wild female, which presumably
has 56 as the oogonial number, and in the fifty or more eggs which
were fixed, there are eight good or fair preparations showing both inner
and outer ecjuatorial plates. In all of these there are certainly not
less than 28 chromosomes in each plate (Fig. 10 a, b). In most 28 are
clearly shown ; in three figures, one plate might be counted as 29.
In the case figured, the outer plate has 27 chromosomes which are
clearly separate, and, in addition, a pair which might be one dividing,
or two at somewhat difi'erent levels. I regard 28 in both plates as
the most probable number, but the double chromosome may perhaps
correspond with the chromosome described by Seiler- which becomes
double in one of the second polar plates, but remains a single chromo-
some in the other. I find no constancy of behaviour in the eight

' J. W. Jenkinson, Qiturt. Joiini. Micr. Science, Vol. XLViii. 1905, p. 407.
- J. Seiler, Zvul. Ameiger, Vol. XLi. 1913, p. 24G.

L. DONCASTER 13

eggs which give clear figures, and am inclined to regard the double
chromosome merely as one which has begun its normal division some-
what in advance of the rest.

I have good figures in the eggs of ten females of the strain with
55 oogonial chromosomes ; in altogether about 37 eggs both inner and
outer plates can be counted, and in some 20 more one or other (usually
the outer) can be counted.

In all the good figures in which the chromosome number in both
inner and outer plates can be determined with certainty, there are 28
in one and 27 in the other (Figs. 11 — 13). In some families there are
about equal numbers of each arrangement, e.g. in brood '13.48 there
are two cases with 28, three with 27, in the outer plate. This family
is known to include males. In other ftimilies there .seems to be con-
siderable excess of eggs which have 27 in the inner plate and 28 in
the outer; e.g. in brood '13.4 B, excluding five cases in which there
is some doubt, there are four clear figui'es with 28 in the outer and
27 in the inner, nine in which either the outer can be counted as 28
or the inner as 27, although the second plate is not countable, and
only two in which the outer spindle has 27, the inner 28. In brood
'13.34 there are three with 28 in the outer, 27 in the inner, one with
27 in the inner and the outer is not countable, and one, which is too
oblique for quite safe determination, which appears to have 28 in the
inner group. In these two families, four females have been reared from
'13.4 B, and the surviving six larvae proved to be female by dissection,
and in '13.34 ten larvae were dissected, all females. There are thus
indications that both broods had a large excess of females, and taken
together they show 17 cases with the unpaired chromosome in the
outer plate against two or three with it in the inner. Since females
of this strain have 55, males 56 chromosomes, it is evident that eggs
with 27 in the inner spindle, and therefore in the mature egg-nucleus,
must be female-determining. It is thus to be expected that broods
which have a great excess of females should show a corresponding
excess of eggs with 27 chromosomes in the inner spindle. I have
not yet been able to find a batch of eggs in which all have 27
in the inner plate, and family '13.4 appears to contradict the results
obtained from '13.4 B and '13.34. Seven females have been hatched
and ten dissected in this family, and yet the eggs show a majority of
cases with 28 chromosomes in the inner plate (twelve with 28 in the
inner plate or 27 in the outer, eight cases of the converse arrangement).
In all three families '13.4, '13.4 B and '13.34, there was considerable

14 Chromosomes ami Sex in Abraxas

mortality among the veiy young larvae, and it is possible that the
excess of females is due not to their being families in which few male
zygotes were formed, but that there has been differential mortality in
favour of the females. For the present, therefore, the question must
be left open, whether all-female families are pioduced by the unpaiied
chromosome going into the first polar nucleus in all the eggs, or whether
a ditferential mortality has some share in their production. Since in
some cases, e.g. brood '12.29, more than two-thirds of the eggs laid
were reared into females (in this case 111 eggs, 77 females, no males)
it seems very improbable that the production of all-female families is
due to early death of the males, and the cytological results from '13.4 B
and '13.34 described above suggest that there are families in which at
least in a high proportion of the eggs the odd chromosome goes into
the polar nucleus, but the proof that all-female families are due to the
extrusion of this chromosome in every egg caimot be regarded as
complete imtil a batch of eggs has been found in which all the inner
plates have 27 and the outer plates 28.

That in some families the unpaired chromosome may be extruded
in all or nearly all the eggs is made more probable by the observations
of Morgan on Phylloxera^. In this genus, and apparently in other
Aphids, the male-producing eggs differ from the female-producing in
the fact that in the former one chromosome is always extruded with the
polar body, so that males have one chromosome less than females. It
seems clear, therefore, that the poles of the maturation spindle are not
necessarily alike, since in Phylloxera and other Aphids a particular
chromosome always goes to one pole. It is not very improbable, there-
fore, that in Abraxas the difference between bisexual and all- female
families consists in the amount of unlikenees between the poles of the
first maturation spindle. Bisexual fixmilies would arise when it is
equally likely that the unpaired chromosome should go to either pole ;
all-fcmale families when it always goes to the outer pole, and families
with great excess of females when it goes much more frequently to the
outer than to the inner.

Another line of evidence with regard to the part played by the
extra chromosome in sex-determination might bo provided if gynan-
dromorphs should appear in families which are known to produce
binucleate eggs. In the only egg of this kind in which it is possible
to count the chromosomes (one of those from '13.42), although the
figures are not very good, each appears to have 27 in the outer spindle
1 T. H. Morgan, Jonrn. Exp. Zool. Vol. xii. 1912, p. 479.

L. DONCASTER 15

and 28 in the inner, which would indicate that both egg nuclei would
be male-determining.

To obtain the complete answer to the questions raised in the
preceding paragraphs will require the examination of many more eggs,
and it is not unlikely that fresh material will be needed. Meanwhile,
one point of great importance appears from the observations made up
to the present time — they show that in Abi-axas, and therefore probably
in other Lepidoptera, the presence of two heterochromosomes is charac-
teristic of the male, and one, of the female. This follows of necessity
from the fact that some eggs have 27, others 28, and that females of
the strain used have 55 and males 56. In strains with 56 in the
oogonia and 28 in all the eggs, it must be concluded that the additional
chromosome is functionless as regards sex, as in the case of other insects
where there is a large and a small heterochromosome in the male. The
Lepidoptera thus show a condition which is the exact converse of that
found in all cases hitherto described. The connexion of this fact with
sex-limited inheritance will be referred to again in the next section.

e. The Exceptional Faiiiilij 12.25.

In my previous communication on the chromosomes of the unisexual
families (Journ. Genet. III. p. 9), I mentioned that one female of family
'12.25 showed an oogonial figure with 56 chromosomes, while others of
the same brood had clearly 55. At that time I had no other clearly
countable figures in females which were daughters of unisexual broods,
but which themselves belonged to bisexual broods, and I therefore
concluded that some females of such broods had 55, others 56. Sub-
sequent work, however, as described above, has shown that with this
one exception, all females of the strain with 55 chromosomes, whether
they belong to unisexual or to bisexual families, have 55. The single
female of brood '12.25 is thus alone in apparently having 56 (cf Figs. 17,
18). Very careful examination, with both 2 mm. and 3 mm. Zeiss
apochromatic lenses, shows that in the single good figure two of the
chromosomes counted separately are in contact, and might conceivably
be dividing halves of one (Fig. 18). This, however, is made improbable
by the fact that one of the halves is itself somewhat divided. Many of
the chromosomes show some extent of doubleness, in preparation for
division, but none of them are so conspicuously double as the chro-
mosome in question, if it is really one in division, and the distinct
doubleness of one of the halves is also inexplicable if the pair are really
halves of one chromosome. The evidence therefore seems to me to be

16 Cliroiiiosomen and Sex in Abnixas

strong that in this figuiv there arc in f.ict 56 chromosomes in place
of the normal 55. I should not lay any great emphasis on a single
exceptional case in which only one equatorial plate is available, if it had
nc)t appeared, after the record of the exceptional count was published,
that family '12.25 was peculiar in another respect. The chroinosomc^s
were counted in the winter of 1912-13 ; when the moths hatched
in the following May and June, two females of this family, out of a
total of 16 reared, were ijrossulariata instead of lacticulor. The ])arents
were gruns. $ x lact. ,} ; the normal result of such a mating, owing to
the sex-limited transmission of the (jvu^s. character by the female, is
that all male offspring should be tjross., all females lact. The males
were all f/ro.ss. (21 were reared), but the presence of two gross, females
shows that in this case there was an exception to the normal sex-limited
transmission.

I have very rarely had such excej)tions in previous years, but
since the moth is very common, and the possibility of the accidental
introduction of a wild larva with the food-plant difficult to guard
against with absolute certainty, I have always hitherto regarded such
exceptions as at least possibly due to accident rather than to failure
of the normal sex-limited transmission. In tiie present case, however,
such an explanation is impossible. The two exceptional females are
very closely alike (Figs. B, C), and show peculiarities in their markings
which make it certain, not only that they are sisters, but also that
they are daughters of the mother of brood '12.25 (Fig. A). Their
peculiar features are quite exceptional in wild (/rutmalariata (cf Fig. E),
but exist in the mother and other grossulariata ancestors of the family.
Some of these features, it is true, are found in collaterally related
families (Fig. D), and it might be argued that two larvae had been
accidentally transferred from another brood. Apart from the great
improbability of such an accident in carefully controlled experiments,
I have no other specimen which combines the peculiarities in the way
seen in the females in question, and it seems certain that they are
genuine exceptions. Similar exceptions, of course, are well known
in other species, e.g. Canaries (Miss Durham) and Pigeons (Staples
Browne).

Now it is a very remarkable coincidence, if it be coincidence, that
the only exception to the number 55 in the oogonial chromosomes
should occur in the same brood as these two very rare exceptions to the
normal sex-limited transmission. If, however, it is not coincidence, the
facts fall quite simply into line. In the strain with 55 chromosomes,

L. DONCASTER

17

it has been shown that eggs with 27, fertilized by sperm with 28,
give rise to females with 55, while eggs with 28 give rise to males
having 56. Since the rjrossidariata factor is normally transmitted only
by the male-producing eggs, it must be supjaosed to be associated with
the extra chromosome in the male-determining egg. If, however, the
male factor M and the gross, factor G are not borne in one indivisible
chromosome, but in a compound chromosome (such as has been de-
scribed by Seller in eggs of the moth Phvarjniatobiu), and if in these
exceptional cases the compound chromosome separated into its com-
ponents, so that the G portion remained in the egg and the M portion
went into the polar nucleus, an egg would then be produced which was
female-determining, bore G, and had 28 chromosomes. Such an egg
when fertilized by a lacticolor male would give rise to a r/rossulariuta
female with 56 chromosomes, instead of the normal result, namely, a
lacticolor female with 55. Diagrams will make this clearer.

Gross. ? with .55

Eggs

Offspring

A. Normal Case.

.'54 + il/G 5ih1Mu lact. male with ,56

27 + JI/17 27 + il/(/ spermatozoa

5i + Mg

lact. female

55 chromosomes

54 + MG + Mg
gross, male
56 chromosomes

Gross, s with 55

Eggs

B. Exceptional Case.
5i + 31G 54 + 2J/3 lact. male with 56

54 + ,% -tG

gross, female

56 chromosomes

Journ. of Gen. iv

27 + G 27 + jI/ 27 + il/y •n + 3Icj spermatozoa

bi + M+Mg

lact. male

56 chromosomes

18 ('/int/ii,o,soinefi and Sex in Abraxas

Uii this hypothesis, there should of course be ;is many exceptional
lacticolor males as (jrossulariata females, unless, as is possibli', when ^1/
and G separate, M always goes into the polar nucleus. In any case,
since only 21 males were reared, the absence of lacticolor males from
the family is not surprising'.

Since only one oogonial figure with 56 was found, and this is not
entirely unequivocal, it is obviously impossible to found any important
theoretical conclusions on the case, but the fact that the abnormal
chromosome number was recorded before the existence of exceptional
transmission of the ijros.'i. character was suspected, makes some discu.ssion
of the matter justifiable.

It should be mentioned that two other exceptions occurred in the
moths hatched in 1913, for which I can offt'r no explanation. In family
'12.40, of which the parents were lacticolor $ and wild tjru.ssulariata </,
among 19 gross, males and 14 gross, females, there were one male and
one female lacticolor'-.

Summary.

1. The families reared in the year 1912-13 in general confirm the
conclusions given in the former paper on the inheritance of a tendency
to produce all-female amilies in Abraxas grossidariata. About half
the females belonging to such families usually have only female off-
spring, but the 1912 inatings show that they may sometimes have a
few males amctng a very lai'ge excess of females. The tendency to
produce only females is thus variable in intensity, and ranges through
varying degrees from equality of the sexes to complete absence of males.
In families in which males are produced, evidence is given that there is
no important difference in the sex-ratio between the offspring of the
earlier and later eggs laid by the mother. When there is great excess
of females, there appears to be a tendency for this excess to diminish

' Since this was written, C. B. Bridges has published a hypothesis to account for excep-
tions to sex-limited transmission in Drosopliila. (Journ. Exp. Zool. Vol. xv. 1913, p. 587.)
He suggests that exceptionally the sex-chromosomes may fail to separate at the maturation
division, and both pass into one gamete, so that gametes with two sex-chromosomes, and
others with none, would be provided. If a spermatozoon with no MG chromosome
fertilized an egg with one, a iirost:idariutu female would result. In this connexion it may
be noted that one primary spermatocyte with only 27 chromosomes was found in a male
of '12.7, the father of which was brother to the male parent of '12.25.

2 A female hictirolor could arise from this mating from a spermatozoon lacking the
sex-chromosome, according to Bridges' hypothesis mentioned in the footnote above, but it
is impossible on this hypothesis to account for a male lavticolor in this case, unless
'non-disjunction' occurred simultaneously in the egg.

L. DONCASTER 19

in subsequent generations. Sterility is not much more frequent in
all-female fiiniilics than in normal families.

2. In counts of mitotic figures in ovaries of more than fifty females
descended from unisexual families, 55 chromosomes were found in every
case (with one exception in family '12.25 ; see No. 5 below). About
twenty of these figures wore so good that doubt about the number is
hardly possible, and in several the number 55 is absolutely certain. The
number 55 was also counted in oogonia of one wild female, but in three
other wild females and in four pedigree individuals not descended from
the all-female producing stock there were clearly 56.

3. Males, whether of the stock in which the female has 55 or 56,
always have 56 spermatogonial and 28 secondary spermatocyte chromo-
somes. In some cases, more commonly in the stock in which the female
has 56 oogonial chromosomes than in that in which it has 55, two
chromosomes in the secondary spermatocytes are in contact, but it is
almost certain that there are never really 27. A single figure with
27 in the first spermatocyte was found, but many others in the same
testis had 28.

4. Both equatorial plates of the second polar mitosis in eggs of
females which have 56 oogonial chromosomes, have 28. Eggs of females
with 55 in the oogonia have 28 chromosomes in one polar equatorial
plate, 27 in the other. In some families it is equally common to find
27 in the inner or the outer plate, and some of these are known to be
bisexual families. In two, however, there is a considerable excess of
eggs with 27 in the inner plate, and these are known to be families
with excess of females. In one family, in which females are in excess,
a majority of the figures counted show 28 in the inner plate. It is
suggested that the excess of females in this family is due to mortality
of the males. The facts as a whole make it clear that eggs which
eliminate the 28th chromosome become females, those which retain it,
males; Abraxas thus shows a condition which is the converse'of that
described in most other insects.

5. In the previous paper it was recorded that one female of family
'12.25 had 56 chromosomes (only one figure can be counted). Other
females of the same brood have clearly 55. In the same brood there
was failure of sex-limited transmission of the grossuhmata character
in two cases, in such a way that the grossuhmata mother transmitted
this character to two of her daughters (out of a total of 16), instead of,
as normally happens, only to her sons. It is suggested that this may

2—2

20 ClironioHomcs and Sex in Abraxas ■

be correlated with the extra chroincisdine fnuiid in one female of this
family, the (jrossidoriata bearing chromosome having become separated
abnormally from the scx-chromosomc.

6. In the section on the maturation of the egg three cases are
described of eggs which have two nuclei, both undergoing maturation
simultaneously. In each case the two maturation figures are in exactly
the same stage ; in two they are in the equatorial plate stage of the
second polar mitosis, in the third egg both figures show three polar luiclei
and conjugation of the egg-nuclei with sperm-nuclei. It is suggested
that such an egg, if it had developed, might have given rise to a
bilateral gynandromorph, and th;it such gynandromoi-phs perhaps arise
in this way. In the egg in which there are two pairs of conjugating
pronuclei, the sperm -radiations are sinuous and branch, and those of
one system cross those of the other.

Cambbidoe,

January, 1911.

EXPLANATION OF PLATES.

All the microscopic tif^ures, witli the exception of No. 18, were drawn with Zeiss 3 mm.
apochromat. objective, n. ap. IIO, and with compens. ocular 12. The drawings are free-
hand, and are not all on exactly the same scale.

PLATE I.

Fig. 1. Wild ■? . Oogoiiial eijuatorial plate, .')6 chromosomes.

Fig- 2. „ „ „ 55

Fig. 3. ? of '12.18. „ ,, 55

Fig. 4. Another ? of '12.18. Equatorial plate of cell in synapsis zone of ovary,

55 chromosomes.
Fig. 5. ? of '12.31. Enuatiirial )ilate of cell in synapsis zone of ovary, 55 chromosomes.
Fig.fi. Same ? of '12.31. Oogonial equatorial plate, 55 chromosomes.
Fig. 7. ? of '12. 2U. Oogonial equatorial plate, .55 chromosomes, the double chromosome

marked x is almost certainly one dividing.
Fig. 8. ? of '12.21. Oogonial equatorial plate. The small rings above the chromosome

group indicate stained bodies which are certainly not chromosomes.
Fig. 9. i of '12.18. Spermatogonial plate with 50 (cf. Figs. 3, 4, oogonial plates with .55

from females of the same brood).
Fig. 10. a, outer; h, inner equatorial plate in egg of a wild ?. 28 chromosomes in

each plate.
Fig. 11. Egg of '13.4. a, outer polar plate with 28; b, inner with 27 chromosomes.
Fig. 12. Egg of '13.34. », outer polar plate, with 27 in one section and one lagging

chromosome (separated from the rest by a line) in the next -section; h, inner polar

plate with 27.
Fig. 13. Egg of '13.48. a, outer polar plate with 27 ; h, inner with 28.

L. DONCASTER 21

PLATE 2.

Fig. 14. Egg of '13.3. Secoud polar mitoses, side view, to show the appearance resembling
cilia. Combined from two successive sections.

Fig. 15. Egg of '13.4. Showing two maturation figures in one egg. The two parts of
the figure are drawn from sections five sections (50;n) apart.

Figs. 16a, 16b. Egg of "13.42, with two maturation figures in one egg.

Fig. 16a. Combined from two successive sections, showing three polar nuclei, the two
inner in contact, in each maturation figure. The outer nuclei of both figures are in
one section, the two inner pairs in the next.

Fig. 16h. Conjugation of the two female pronuclei with two sperm-nuclei. Combined
from the two sections next following the sections from which 16(( was drawn. Note
that the sperm-radiations are sinuou.s, bifurcate, and those of one system cross those
of the other. The clear space around the conjugating nuclei is due to contraction at
fixation.

Fig. 17. ? of '12. '25. Oogonial equatorial plate with 5.5 chromosomes. (Only 54
are visible in the figure ; one has been accidentally omitted.)

Fig. 18. Another ? of '12.25. Oogonial equatorial plate with 56 (exceptional). The
only doubt about the number 56 arises from the fact that the two chromosomes
marked x might possibly be one in division. Since the outer is itself double, this is
very improbable. Drawn with Zeiss 2 mm. apochrom. n. ap. 1'40, compens. oc. 12.

PLATE 3.

Fig. A. Female parent of brood '12.25.

Figs. B, C. The two grossulariata females which appeared as exceptions to the normal
sex-hmited transmission of the gross, character, in brood '12.25. Note the close
resemblance of the pattern in the two specimeus, and their general resemblance
to their mother. The chief peculiarities are

(a) the union of the costal and discal spots to form a definite hook-shaped or
?-3haped mark across the middle of the fore-wing ;

(h) the reduction, but not complete absence, of the spots at the tip of the fore-
wing on the outer side of the yellow band ;

(c) the union of the basal line of spots on the hind wing into a continuous band,
from which the discoidal spot projects outwards, but is continuous with the band ;

(d) the almost complete union of the distal row of spots on the hind wing, and
the absence of the most anterior marginal spot, which ia usually present at the costal
angle.

These points are indicated by letters in Fig. C.
Fig. D. A grossulariata female of brood '12.15, closely related to '12.25. This is the

specimen most nearly resembling the gross, females of '12.25 among the moths bred

in 1912.
Fig. E. Atypical grossulariata female; showing the difference between the females of

'12.25 and ordinary grossulariata.

JOURNAL OF GENETICS, VOL. IV. NO. 1

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JOURNAL OF GENETICS, VOL, IV. NO. 1

PLATE III

*'^— ' ^Jlff

^••^

ON INHERITANCE OF WEIGHT IN POULTRY.

By R. C. PUNNETT, M.A., F.R.S.,
AND P. G. BAILEY, M.A,

CONTENTS.

PAOE

Introduction . 23

The Hamburgh- Sebright Cross 25

The F-i generation 27

The F.| generation 29

The Hamburgh- Sebright x Brown Leghorn Cross .... 31

The Hamburgh-Sebrightx (Bantam X White Leghorn) ... 31

Hypothetical Explanation 33

Explanation of Plate 39

Introduction.

Few experiments have been undertaken with the object of investi-
gating the inheritance of size or weight in animals. Beyond the
observations of Goldschmidt' and of Phillips- on ducks, and of Castle^
on rabbits practically nothing definite is known. Nor are all of these
observations sufficiently extensive to advance our knowledge greatly.
Goldschmidt only reared an F.. generation from one cross and that
consisted of but 8 individuals. Moreover as his weight records cease
at the age of 10 weeks little is to be learned from them that bears
upon our present enquiry. Castle's work on the weights of rabbits is
too scanty to give much definite information. Phillips' experiments
however are more extensive and suggestive. From the cross Mallard x
Rouen he reared 13 F^ and 33 ^2 birds. The Fi birds were inter-
mediate in size as compared with the parents. The mean of the
F„ birds was also near that of the Fi, but their range of variation
was considerably greater. None however were as heavy as the Rouen,
nor were any as light as the Mallard. The numbers however are too
small to be able to state definitely that these classes do not appear in

1 Zelt.f. iiid. Abst. Vererb. 1913. 2 Journ. Exp. Zool. 1912; ibid. 1914.

'■> Publ. Cameij. Inst., Wash., No. 114, 1909.

24 On Inheritance of Weight in Po^iltry

F«. Nevertheless the great i-ange of variation in F.2 suggests an
interpretation by means of several segregating factors, though this
cannot be decided definitely without raising an F^ generation. Whether
size in animals can be expressed in terms of Mendelian foctors is there-
fore still an open question.

The present paper gives an account of some experiments which we
have been carrying out during the past few years with the object of
learning something about the transmission of weight in poultry. For
this purpose two breeds were selected which differed considerably in
size, but not sufficiently to prevent natural crossing. At the same time
it was considered advisable for reasons of economy that the larger breed
used should not be too large ; otherwise it would not have been possible
to rear so many oHspring to maturity. The two breeds eventually
selected were the Gold-pencilled Hamburgh and the Silver Sebright
Bantam. The choice was determined with the idea of following up the
inheritance of other characters in addition to size, and in this set of
experiments we have been investigating also the inheritance of gold
and silver ground colour, and the peculiar assumption of the hen's
plumage by the cock which is characteristic of the Sebright. An
account of these characters and their inheritance will be given later.
For the present we shall confine ourselves to what we have been able to
learn from this cross with regard to the transmission of size. More
correctly perhaps we ought to .say weight, since our estimation of size
depends entirely on the weight of the biid. Generally speaking the
bird that looks heavier will be found actually heavier when weighed, but
it is not always possible to be certain from appearance alone. A taller
but more slender and "leggy" bird will sometimes be fovmd to weigh less
than what appears to be a smaller tliough better furnished one. Weight
is doubtless a character which dejjends in some degree on structural
characters capable of analysis. We have not found it practicable to at-
tempt such analysis for the present. Our criterion throughout has been
the weighing pan alone. Observations start with the egg weight. The
newly-hatched chicken is also weighed, and a record is then taken weekly
or fortnightly up to the end of the 35th week. It would of course be
desirable to have a complete record of every bird from the day of hatching
to the day of natural death. In practice this is not possible owing to the
difficulty of keeping so many birds. A bird, especially a cock, may go
on growing more than a year after it is hatched. But we have found
that such growth, when it occurs, is intermittent. Birds hatched from
February to May receive a check in their growth at the end of the year,

R 0. PUNNETT AND P. Gr. BaILBY 25

if not before, and the weight reached by them at the age of 35 weeks is
not exceeded for several months. Later on the bird may show a further
period of growth, but after it has reached the age of 8 — 9 months, or in
many cases even less, there is a distinct interval of several months
during which the weight remains nearly constant. This is certainly
true for the breeds with which we have been working, and we have
consequently kept and recorded the weights of the birds bred up to
35 weeks, after which the majority have been killed. When we speak
of the weight of a bird we mean this weight of the first year's gi'owth
unless the contrary is stated. We may add that all our birds were kept
together on the same land and were fed and treated, so far as possible,
precisely in the same way.

The Hamhwrgh-Sebright Gross.

The original stock with which the experiments were started in 1910
consisted of a trio of Gold-pencilled Hamburghs and 2 Silver Sebright
hens. The Hamburgh (/ when 3 years old weighed 1540 grams; when
35 weeks old it is likely that he would have been some 150 — 200 gxams
lighter. The Sebright $ $ weighed 570 and 620 gi-ams respectively.
We have no record of the weight of a Silver Sebright cock of this
strain but he would probably have weighed about 750 grams. During
the course of our experiments we also used some Gold Sebrights.
These were procured from a different source and were markedly smaller
than the Silvers, a cock weighing but 570 grams and 3 hens 455, 455,
and 480 grams respectively. We raised a few birds from our original
Hamburgh (/ with one of the Hamburgh hens, viz. 1 ^ and 4 $ ^ . In
both sexes these birds were rather larger than the F^ ex Sebright x
Hamburgh (cf Table I, p. 26). We subsequently mated up a brother
and sister from these pure Hamburghs. The offspring were distinctly
smaller and throughout their growth were sickly looking birds. Whether
due to close inbreeding or to some other cause the phenomenon was
very marked, and we have not made use of observations on these birds
in our account. With healthy birds the weight of the Hamburgh is
nearly double that of the Sebright.

The original cross was made between the Silver Sebright hen and
the Hamburgh cock (cf. PI. IV, fig. 1). From it two cockerels were
reared in 1910, and in the following year 8 (/J" and 7 $ ^ were raised.
With regard to size both sexes were fairly uniform. At 35 weeks old
the (/'(/' ranged from 1140 to 1360 grams, while at the same age the
$ % were between 940 and 1110 grams (cf Table I).

2G

On Inheritance of WeUjIil in PonJfri/

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R. C. Pdnnett and p. Q. Bailey

27

In general build and habit the -^i $ $ were all remarkably alike
(cf. PL IV, fig. 3). Of the F^ cocks there were two classes, viz. those with
henny plumage (PI. IV, fig. 2), and those with normal plumage. Within
each class however the individual members were very similar to one
another. The difference in plumage was not found to be correlated
with any size difference either in Fi or in subsequent generations. The
Fi birds then from this cross were rather smaller than the pure Ham-
burghs, though of course very much larger than the Sebrights. Of
these Fi birds 13 were bred from, viz. 1 J' J' and 6 $ $. Most of them
were mated with one another but 2 (/J' were run respectively with
Gold Sebright $ $ (p. 30) and with 2 i^, $ $ fi-om a bantam x White
Leghorn cross (p. 31). The figure below provides a concise scheme of
the experiments.

Sebright ? x Hamburgh i

I

, , . , 1 ,

Fii X Fi i

(Pens 21- 25,

1912—13)

F2t X F2S

FiS X Gold Sebright ? ?
(Pen 7, 1911)

i X S

FiS x2Fi'i $ (Bantam x

White-Leghorn)

Pen 4, 1911

? X <?

Pen 12,

Pen 13,

Pen 7,

Pen 1, Pen 2,

Pen 14,

Pen 4

1913

1913

1913

1912 1912

? X <J

Pen 20, 1913

1912

1912

The Fo generation. During 1912 and 1913 five pens of Fi birds were
bred from. Each of these contained a single pair, except that in 1912
one of the F-i cocks was run with two of his sisters. We have analysed
the results given by each pen separately, but they are very imiforni,
and as we can detect no significant difference we shall consider them
together. In all we have raised 233 F„ birds to maturity, viz. 112 J" jf
and 121 %%■ In both sexes the range of size is very considerable.
The {/(/ vary from 680 — 1420 grams while the pullets weigh from
540 — 1290 grams. Allowing for sexual difference the range of variation
is evidently similar in the two cases, and in each of them the weight of
the largest bird is distinctly more than double the weight of the smallest.
For each sex also the range of variation is such that the smallest birds
are smaller than the Silver Sebright bantam, while the largest are larger
than the Gold Hamburgh.

28

On Inheritance of Wen/ht in Poulfri/

-I 1 1 1 1 1 1 r

700 900 1100 1300

800 1 000 1 200 1 400

^2 males, 1912 and 1913.

^°°600^°%00^°°1000"°°1200'3°0
F.2 females, 1912 and 1913.

^°%00^°°1000"°°l200l^°°l400
F-i males, 1912.

1 1 I I I r" r
500 ^„„ 700 „„ 900 „ „1100 1300

600 800 1000 1200

F; females, 1912.

^°°800^°°1000^^°°1200^3°°1400

^°°600'°%00^°°1000"°°1200

F2 males, 1913. P2 females, 1913.

Fig. 1. Weight curves of F^ birds at 3-5 weeks.

R. C. PUNNETT AND P. Gr. BaiLEY 20

In Fig. 1 the F^ weights at intervals of 50 grams have been
arranged to give the curves of variation \ For each sex these curves
are irregular. Probably they cannot be regarded as simple curves of
error, but appear rather to suggest that we are dealing in either instance
with compound curves made up of several simple ones. Further
analysis however must come from the breeding pen, and in this direc-
tion we have already taken the first step with some experiments in
raising a further generation from certain of the F. birds.

Tlie F3 generation. It was clear that the first step towards analysing
the F., birds was to test whether the largest and the smallest bred true.
With this purpose three pens of F^ birds were mated up in 1913, viz. a
pair of the largest birds and two jaairs from among the smallest.

(A) For the pen of larger birds we selected an F. hen (PI. IV, fig. 6)
which was more than 100 grams heavier than any other F.. $ yet bred.
Her weight at 35 weeks was 1290 grams. The F„ ^ chosen (PI. IV, fig.
5) weighed 1390 grams at the same age, and was among the heaviest
of the 1912 birds. (In 1913, however, we bred two heavier F„ cockerels.)
From this mating 13 (/(/ and 13 $ $ were reared during the past
season. The </(/ ranged between 990 and 1630 grams, averaging
1360 grams, while the % % were between 930 and 1350, averaging
1150 grams. The offspring of the large F^_ birds must all be reckoned
on large side, though they showed a good deal of variation. Nothing
approaching a bantam was jjroduced by these two birds.

(B) Of the two pens of small F„ birds put up in 1913 one (Pen 13)
consisted of a pair of the smallest F« birds reared by us. The </ (PI. IV,
fig. 5) weighed 680 grams and the % (PI. IV, fig. 6) but 540 grams. These
figures are rather below the average weights of the original Sebrights.
Unfortunately the hen died before she had laid many eggs and we only
reared five birds from her. Of these 2 were ^^ and 3 were % % , and
all of them were below 600 grams.

(C) The other pair of small F„ birds was rather larger than that
just discu.ssed. Here (Pen 7, 1913) the </ weighed 740 and the ^
710 grams. She must probably be regarded as belonging to the
smaller of the intermediates, rather than as pure extracted bantam.

' The curves for the two years 1!)12 and 11113 have been given separately. The
average weight of the birds in the latter year is somewhat lower than in the earlier one.
This is due to an outbreak of diphtheritic roup which affected about half of the F^ birds
in 1913. Many of these birds had probably not recovered their normal weight when killed
at the age of 35 weeks.

•')() On Inheritance of Weif/ht in Poultrij

Fn.iii iJiis mating wrrc rcari'd (i </(/ and 7 $$. The (i </(/ rangrd
h(jin 720 to 8(i0 grams, averaging 800, half being about the same size
as the father and half rather larger. Of the 7 ^ $ four were smaller
than (,heir mother while the other three were of about the same size or a
little larger. In other words there were nearly eijual numbers in l)oth
se.\es of regular bantams and (jf biids rather larger. The residts ai-e in
accord with the view that the father was a true bantam, and that the
mother was heterozygous for a factor which had the effect of increasing
the weight of a bantam by about 25 7o-

These F^ results taken together suggest stnjngly tha,t size in poultry
depends upon definite factors, and that these factors segregate in
gametogenesis. More experimental data are required before we can
hope to determine the nature anci number of these factors, and we have
already designed some further experiments with this end in view.
Meanwhile we may give an account of some other data. \vi' havt' got
together in connection with the inheritance of size.

i^i ,/ X Bantam. We mentioned above that two i*', ^ ^ ex Silver
Sebright x Gold Hamburgh were reared in 1910, but that owing to
the lack ol F^ $ $ we were unable to breed an F. generation in the
following year. One of these F, J ^^ was put to two Gold Sebright
hens in 191 1 (Pen 7, imi ) and from this mating 7 c/j" and 11 ? ? were
reared. Of the former 2 were bantams under 700 grams, while the
remaining 5 ranged from 800—900 up to 1100—1200. Of the pullets
6 were undei- 700 grams, 3 were of the usual F^ size and the remaining
2 were intermediates. From these birds two pens, 1 and 2, were mated
up in 1912.

Pen 2, 1912. ^ 31(3 (weight 620 gr.) x % 283 (weight 620 gr.).
This pen gave 7 {/•(/ ranging between 680 and 880, and 8 pullets all
under 600. These birds were evidently breeding nearly true to small
size. In the following year the strain was further tested by mating
together two of these 1912 birds from Pen 2. They were ^ 232 (weight
690 gr.) and ? 678 (weight 470 gr.). They produced in 1913 10 j",/
between 540 and 750 grams, and 3 $ $ all under GOO.

The experiment shows that the original F^ j" ex Sebright x Ham-
burgh must have been producing some "bantam" gametes. Such
gametes, meeting with the gametes carried by pure bantams, gave rise
to birds which belonged to and bred true to the small bantam size.

Pen 1, 1912. The other pen mated up with birds from the F^ x
Sebright cross consisted of a cock and 2 hens. The former was rather

R C. PUNNETT AND P. G. BaILEY 31

above bantam size, weighing 825 grams, while the hens were respectively
620 and 650 grams. The progeny showed a good deal of variation (e.g.
Table I). A few were of the smallest bantam size, others were near
the parents, while a few were distinctly larger. Two of the cockerels
for instance were between 900 and 1000 gi-ams while one of the pullets
was over 800. The result shows that birds which are not quite of
bantam size may give both birds distinctly larger and others as distinctly
smaller than themselves.

The Hamburgh Sebright x Bruvm Legliorn. Cross.

These experiments were made primarily with the object of following
up the transmission of the female form of plumage that may occur in
cocks. They offer also some interesting data in connection with the
inheritance of size which may be considered here. The ^ J' alone were
reared, and as they generally show their type of feathering at 6 months
old they were for the most part killed at that age. Had we been able
to keep them for a further 3 months it is probable that the larger birds
would have grown relatively more than the smaller ones. With Brown
Leghorn hens were crossed F^ ^ ^ and two large F„ ^ ^ (Nos. 28 and
139 of 1912) whose weights, 1200 and 1250 grams respectively, were
those of i^i birds. A few birds were also raised from an i^, % mated
with a Brown Leghorn ^. Similar results were obtained in all of the
above cases (cf Table I) and they may conveniently be considered
together. The figures have been put into the form of a curve in Fig. 2
(on p. 32). It is evidently bimodal. What is of peculiar interest is that
one of the modes, the higher, jDractically coincides with that of the pure
Brown Leghorn, while the lower one is the same as the single mode
on the curve got from the crossing of Brown Leghorn ,/ with two small
F« $ ^ . The bearing of this will be considered later.

Hamburgh-Sebright x Bantam- Leghorn.

We have already mentioned that in 1910 we failed to breed any
F^ %% from the Hamburgh-Sebright cross. Of the two F., ^ ^ bred
that year one was put to Gold Sebright $ % as stated above, and
the other was run with two hens which had resulted from an artificial
cross between a White Leghorn and a bantam \ They were nearly of
the same weight, one being 1020 and the other 1040 grams. 29 birds
were produced from the pen (Pen 4, 1911), viz. 16 (/'(/ ranging from

' These two hens were given to Professor Bateson in 1908 by Dr Hagedoorn, who
stated that they were made by artiticially inseminating a bantam ben with the sperm of a
White Leghorn cock. The nature of the bantam used is unknown.

32

On Inheritance of Weight in Poultrij

1000 1100 1200 1300 1400 1500 IGOO 1700 1800 1900
Fig. 2. Weights of Hamburgh-Sebright x Brown Leghorn Crosses at 24 weeks.

A. Male offspring from small Fo ? ? x Brown Leghorn <J (Pens 14 and 26, 1913).

B. Pure Brown Leghorn j i .

C. Male offspring from ¥x x Brown Leghorn and from two large Fa birds x Brown

Leghorn.

R. C. PUNNETT AND P. G. BaILEY 33

940 up to 1710 grams, and lo $ $ between 765 and 1190 grams. The
wide range of variation accorded with the nature of these two hens sent
by Dr Hagedoorn, and this was confirmed by the following year's work.
The largest and the smallest pair were mated up in 1912. From the
largest pair (Pen 14, 1912) were produced 12 ^/c/ and 7 $ $. The
average weight of the (/</ was not very different from that of the 1911
family, but the 7 $ $ were a distinctly heavier lot (cf. Table I). The
smallest pair of birds (Pen 4, 1912) produced 7 </</ and 5 ^ $. As
the table (p. 26) shows all of these birds were small, the majority being
smaller than the parents. In the case of the pullets the range of varia-
tion of those from the small parents is entirely outside the range of
those from the large parents ; in the case of the cockerels the two
ranges just overlap. These experiments were discontinued after 1912
in favour of the Hamburgh-Sebright cross. The general results how-
ever, the great range of variation in F.;,, and the fact that the extremes
tend to breed true to size, offer an interesting corroboration of the
Hamburgh-Sebright story.

We may now proceed to suggest a scheme by which the phenomena
we have described may be interpreted in terms of genetic factors.

H ypothetical Explanation.

In discussing such a tentative scheme several distinctive features
about the results must be borne closely in mind. Among them the
most important are :

(1) The fact that from the Sebright-Hamburgh cross the range of
variation in F^ is beyond that of either of the original parent forms.
The smallest birds are definitely smaller than the Silver Sebright and
the largest are well above the weight of the Gold Hamburgh. This
phenomenon was more marked in the F^ generation when the smallest
and largest F^ birds were bred fi'om. It must therefore be supposed,
in devising an explanation in terms of genetic factors, that the Gold
Hamburgh does not contain all the factors concerned, and that the Silver
Sebright does not lack them all.

(2) The Fi birds are not quite so large as the Gold Hamburghs.
They must however contain a single dose of all the factors concerned.
Hence we must suppose that the factors with which we are dealing
produce a less marked result when the bird is heterozygous for them
than when it is homozygous.

(3) Extreme variants in F^ are relatively scarce. Indeed the
smallest and largest extremes as shown by the F^ generation did not

Journ. of Gen. iv 3

34 On Inheritance of Weif/hf in Poultvii

occur ill our F.. generation at all. W'c regard this as due to ihr fact
that an insufficient number of birds was bred in F., to give them.
Where F^ birds are heterozygous lor tour factors, the two extremes, i.e.
that homozygous for all four factors and that lacking all four, will only
occur once in 256 birds. The extremes were however readily obtained
in Fs and we have little doubt that we should have obtained theni in F.,
had we been able to breed a sufficiently large number of biixls.

Bearing these points in mind we have constructed a scheme in-
volving the following assumptions.

(rt) That we are dealing with a case in which we are involved with
the presence or absence of four genetic factors each of which affects the
weight of the bird.

(6) That when none of these I'actors are present the birds are of
the minimum size.

(c) That two of these factors, A and B, i)roduce an increase of
66 °/o respectively on the minimum size when the birds are homo-
zygous and 38°/o when the birds are heterozygous for eithe'r of them.

(d) That the remaining two factors, C and D, produce an increase
of 30°/j, respectively when the birds are homozygous, and 25 "/^ when
the birds are heterozygous.

(e) That the constitution of the Gold Hamburgh is AABBCCdd
and of the Silver Sebright aabbccDD.

If we take the minimum weight as 100 for birds of the consti-
tution aabbccdd, the maximum weight for birds of the constitution
AABBCCDD becomes 1 00 -t- 66 + 6(i + 80 + 30 = 292. Hence F, birds
of the constitution AaBbCcDd, themselves of grade 226, should when
bred together give a generation ranging from gi-ade 100 up to grade
292. Since four factors are concerned the extreme members of the
series will only make their appearance in this generation once, on an
average, among 256 birds. Table II shows the proportion and consti-
tution of these 256 birds which go to form the F^ generation. Opposite
the constitution in each case the weight grade is given, and in the
other two columns these theoretical weight grades have been translated
into expected actual weight on the basis of the range of variation as
found by experiment in the F,, and F^ generations. For example the
smallest ^ obtained in Pen 13, 1913, weighed about 550 grams and the
largest bred in Pen 12, 1913, reached a weight of rather over 1600 grams
(cf. Table I, p. 26). In order to obtain the actual weights of the
females as they should be on our scheme we have multiplied the male

K. C. ]'UNNETT AND P. G. BaILEY

TABLE II.

Constitution

Weiclit
grade

\Veiglits

of
Males

Weights
Females

Constitution

Weiglit
grade

Weights
Males

Weights

of
Females

lAABBCCDD

292

1620

1300

SAaBbCcdd

201

1110

890

•2AABBCCDd

287

1580

1270

iAciBbccDD

206

1130

905

lAABBCCdd

262

1440

1150

SAaBba-Dd

201

1110

890

2AABBCcDD

287

1580

1270

iAaBbccdd

176

970

775

iAABBCcDd

282

1550

1240

2AabbGCDD

198

1090

870

2AABBCcdd

257

1415

1130

iAahbCCDd

193

1060

850

lAABBccDD

262

1410

1150

2AabbCCdd

168

925

740

2AABBccDd

257

1415

1130

iAabbCcDD

193

1060

850

lAABBccdd

232

1275

1020

8AabbCcDd

188

1030

825

2AABbCCDD

264

1450

1160

iAabbCcdd

163

900

720

iAABbCCDd

259

1420

1130

2AabbccDD

168

925

740

2AABbCCdd

234

1290

1030

iAabbccItd

163

900

720

iAABbCcDD

259

1420

1130

2Aabbccdd

138

760

610

HAABbCcDd

254

1390

1110

\aaBBCCDD

226

1240

995

iAABbCcdd

229

1260

1010

2aaBBCCDd

221

1215

970

2AABbccDD

234

1290

1030

laaBBCCdd

196

1080

870

iAABbccDd

229

1260

1010

2aaBBCcDD

221

1215

970

2AABbccdd

204

1120

900

iaaBBCcDd

216

1190

950

lAAbbCCDD

226

1240

995

2aaBBCcdd

191

1050

840

•2AAbbCCDd

221

1215

970

laaBBccDD

196

1080

870

lAAbbCCdd

196

1080

870

2aaBBccDd

191

1050

840

2AAbbCcDD

221

1215

970

laaBBccdd

166

916

735

4AAbbCcDd

216

1190

950

2aaBbCCDD

198

1090

870

2AAbbCcdd

191

1050

840

iaaBbCCDd

193

1060

850

lAAbbccDD

196

1080

870

2aaBbCCdd

168

925

740

2AAbbccDd

191

1050

840

iaaBbCcDD

193

1060

850

lAAbbccdd

166

916

735

SaaBbCcDd

188

1030

825

2AaBBCCDD

264

1450

1160

iaaBbCcdd

163

900

720

iAaBBCCDd

259

1420

1130

2aaBbccDD

168

925

740

2AaBBCCdd

234

1290

1030

iaaBbccDd

163

900

720

iAaBBCcDD

259

1420

1130

2aaBbccdd

138

760

610

SAaBBCcDd

254

1390

1110

laabbCCDD

160

880

705

iAaBBCcdd

229

1260

1010

2aabbCCDd

155

855

685

2AaBBccDD

234

1290

1030

laabbCCdd

130

715

570

iAaBBccDd

229

1260

1010

2aabbCcDD

155

855

685

2AaBBccdd

204

1120

900

iaabbCcDd

150

825

660

iAuBbCCDD

236

1290

1030

2aabbCcdd

125

690

550

SAaBbCCDd

231

1270

1020

laabbccDD

130

715

570

iAaBbCCdd

206

1130

905

2aabbccDd

125

690

550

8AaBbCcDD

231

1270

1020

laabbccdd

100

550

440

UAaBbCcDd

226

1240

995

3-2

;><) Oil I nheritaiice of Weiijht in PouUnj

weights by ^, a proportion which experience has shown us to express
very nearly the relation between average weights of the sexes in the
material we have worked with.

In Table III we have interpreted the experimental results in terms
of the scheme just outlined. It is clear from this table that the scheme
covers the facts fairly closely. The chief (liscre[)atice is in I ho figures
for the F.. generation where the j)roportion of heavy birds among the
pullets is somewhat below expectation. Apart from this the actual
results from the various matings, both with regard to mean weight and
range of variation, tally closely with the figures deduced theoretically on
our hypothesis. It is not impossible that an even closer agreement
might be arrived at if the values given to the various factors were
altered'. At this stage however we are more concerned in demonstrating
that an explanation of these phenomena in terms of definite genetic
factors is possible. To determine the action of I'ach factor with pre-
cision would be a long and laborious undertaking, nor do we propose to
pursue the matter so far. But if our hypothesis is a(le(juate we ought
to find birds <if different intermediate sizes breeding true. We should
for example be able to establish a breed of the constitution AAbbccDD
and of weight grade IflK (Table II). Such a grade is exactly inter-
mediate between AABBCCdd and aabbccDD, i.e. between the Cold
Hamburgh and the Silver Sebright. We ought also to be able to
establish another intermediate race of the constitution aabbCCDD
which would breed true to a grade rather above that of the Sebright.
Experiments on these lines are already in progi'ess. Moreover, until we
have succeeded in establishing such sti-ains we cannot know for certain
what amount of variation is due to non-inheritable fluctuations apart
from the operation of definite genetic factors which affect weight. In
the present paper we have not taken such possible fluctuations into
account as there seems no reason why they should not affect all of the
birds similarly.

We alluded above to soiue experiments in which t\ an<l F., birds
from the Sebright-Hamburgh cross weie mated with pure Brown
Leghorns (p. 31), and we pointed out that in the case of the F^ and

1 For the sake of simplicity we have conceived of our factors as acting in each case
directly on a minimum and constant basal weight. But it is of course possible to regard
them as acting also upon the weight resulting from the other factors present. For
example we have supposed that a single dose of D gives an increase of 25 % on the
minimum weight of 100, whether A, B, or C are present or not. But it may be that D
produces a 2.5% increase on whatever is present. And of course it is possible that weight
factors ma}' act upon one another.

R. C. PUNNETT AND P. G. BaILEY

°.<

s

I— I w

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OOC — OOf

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ooct— OOZl
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38 On Inlien'tance of \Vei)/Jit in Fotiltn/

larger F, birds the results coukl be expressed in the form of a biuiodal
curve whereas a uniiiiodal curve was given by the sniallei- F., birds.
These results are readily explained i>n the hypothesis we have just

•

•

•

•

•

•

•

•

•

•

•

1

100

1

100

1
120

1
140

1
160

1

180

205

120

140

160

180

205

230

Fig. 3.

outlined. The gametes produced by an F, bird are of 16 sorts, viz.
ABCD, ABCd, ABcD, ABcd, AbCD. AbCd, AbcD, Abed, aBCD,
aBCd, aBcD, aBcd, abCD, abCd, abcD, abed. With abed gaimtes
such a series would give rise to the giades 226, 201(2), 176, 188(2),
163(4), 138(2), 150, 125(2), 100. These grades as .shown graphically
in Fig. 3 give rise to a bimodal curve in which the greater mass is
collected on the upper mode. It is the form of curve which we
should expect when Fi or similarly constituted birds are crossed with
some mufurm strain. It is the form of curve which was actually
obtained when F^ (and similar F.,) birds were ero.ssed with the pure
Brown Leghorn (cf Fig. 2, p. 32). Tlic' rcsulls nftci- an interesting
corriiburation of our views as U> I lit- ii.it iiii' of tin- F, birds frniii the
Sebright-Hamburgh cross.

In connection with nur iiiti-rpii'taliiiii we may call attention to tiie
following point of theoretical, and possibly also of practical interest.
On our hypothesis two strains of intermediate and similar weight are
possible, viz. AAbbCCdd and aaBBceDD. A cross between two such
strains would I'esult in F^ biids rather larger than either, while the F.,
generation would be like that shown in Table II. In other words a
cross between two medium sized strains of the same average size may
lead in F., to the production of strains considerably lai-ger and also con-
sideiably smaller than either of the parent strains, both of which can be
readily fixed. It is possible that the great size of many Horal varieties,
e cr. in daffodils, has been effected on these lines from crosses between
medium sized forms differing in constitution for the factors affecting
size. It is not impossible that the increase sometimes observed on

JOURNAL OF GENETICS, VOL. IV. NO. 1

(iolJ-ijeiiciUed HamLimgh J.

Fig. 1.

bllvfl Seliligllt ? .

Liuld-iii'iieilk'd HaiiibuiKli S.

%

-Ji

b\ i (kx Sebunlit ■ lliuiibiiigh).

Fi-. 2.

4-^-

Gold-pLUciUcd Hiimbiivgh J.

l\ f (ex Sebright x Iliimlmigli).

Fig. A.

PLATE IV

V,

if - L

Silver Sebii.''ht ? .

!■', ? (ex Sebriylit x Hamburgh;
FiK. 4.

Small !■:. ■! . (In Pen i;'., 1013.) Large F., i. (In Pen IL', l;il::i.)

Fig. 5.

Small ¥., $. (In Pen 13, 1!)13.) Large F., ?. (In Pen Pi, I'.llS.;

Fig. 0.

R. C. PUNNETT AND P. G. BaILBY 39

crossing strains of animals or plants of similar size is not due to in-
creased vigour resulting from the cross, but to the bringing together
of independent gi-o\vth factors each capable of producing some effect.
Were this the case it would be apparent in the Fo generation where
fixable strains both larger and smaller than the parent forms should
make theii' appearance.

In conclusion we may once more state that the scheme which we
have put forward is a tentative one and may have to be recast in any or
all of its details in the light of futuiv knowledge. Nevertheless there
can be little doubt that it expresses an essential truth in connection
with the inheritance of the complex character designated weight. The
facts of breeding offer a clear indication that weight may depend upon
the presence or absence of definite genetic factors segTegating from one
another in sametooenesis on lines with which students of these matters
are already familiar.

The experiments of which an account is given above form part of a
series of investigations on heredity in poidtry for which the means have
been provided out of the Fund controlled by the Development Com-
mission.

EXPLANATION OF PLATE IV.

In all six figures the birds are taken to the same scale.

Fig. 1. Gold Pencilled Hamburgh s and Silver Sebright ? .

Fig. 2. ,, ,, I? and t'l s ex Silver Sebright x Gold Hamburgh.

Fig. 3. ,, ,, ? and Fi ? ,, „

Fig. 4. Silver Sebright ? and h\ ? ex Silver Sebright x Gold Hamburgh.

Fig. 5. Small FoS (Pen 13, 1913) and Large F.,.l (Pen 12, 1913).

Fig. 6. „ F.f ( „ ) „ t\-i ( „ )■

A PRELIMINARY NOTE ON THE FACTORS CON-
TROLLING THE GINNING PERCENT OF INDIAN

COTTONS.

By H. M. LEAKE, M.A.,

Economic Botanist to Government, United Provinces, India.

The cotton crop, as it is harvested, consists of the lint in which the
seed is embedded. This seed-cotton is then ginned, by whicli process
the seed and lint are separated. The ginning percent of a cotton, as
the term is here used, may be defined as the nmnber of pounds of lint
obtained ft-om 100 lbs. of seed-cotton.

Now the area under a given crop is largely controlled by the price
the actual cultivator receives for his produce. In the case of cotton, the
cultivator parts with the seed-cotton for which the price paid by the
purchaser is almost directly proportional to the ginning percent as
determined by sampling'. The character, which forms the subject of
the present note, is, consequently, one of great economic importance,
and for this reason has formed the basis of a considerable series of
experiments. From the scientific aspect the problem is an attempt to
resolve a complex character into its simpler component iactors.

The range of variation fovuid in the ginning percent of the different
types of cotton at present cultivated may be usefully indicated. In the
crop at present in general cultivation in the United Provinces — consisting
of a mixture of various types of G. iieglectum Tod. — the ginning percent
is 30 — 33. Improved forms, isolated by selection, are now being intro-
duced for which this figure approaches 41. Races of G. cernuum Tod.
have been cultivated in which the ginning percent is as high as 44 or
4.5. At the other extreme lie G. indicum Lamk. and G. arhoreum Linn.
sp. PI. with a ginning percent of 25 — 26, the former giving the Bani
cotton of Central India, perhaps the best indigenous cotton of India, and

' See Leake and Parr, Agri. Jouiii. of IiidUi, Vol. viii. 1.

42 Oinning Percent of Indian Cottons

the latter being the sacred perennial cotton only found now in the
vicinity of Hindu temples. Lastly G. intentiediuiii Ganimie, a tiinii
cultivated as a mixed crop round Allahabad and the West of Bengal, has
a ginning percent as low as 15.

A brief consideration is sufficient to indicate that the ginning percent
is not a simple character. It is directly dependent on the weight of
seed and weight of lint. In {)ractice the fact becomes still more evident ;
thus, among the otfsjjring of nniiierous crosses which have been made
between two parent types, each having a gimiing percent of 25 — 26,
forms have been isolated for which the figures diverge as much as
36 and 18.

The direction of the present investigation will be most profitably
indicated by a short discussion of the a priori considerations on which it
was based. From the definition it is clear that the exact figm-e for the
ginning percent is dependent on the weight of two distinct bodies — the
lint and the seed. This weight is, in each case, dependent on the values
of several characters which, in their turn, may vary. Thus, in the case
of the seed, the weight depends im the volume, on the specific gravity,
and on the number of seeds. In the case of the fibre the characters
affecting tiie weight are not so obvious. They may, howe\er, be con-
sidered as the weight of the individual fibres — clearly not a unit
character as this in its turn depends on such conditions as lengtii,
thickness (mean diameter), size of lumen and specific gravity of fibu-
wall — and the number of fibres. The relation between the number of
seeds and number of fibres in a given .sample i.s again determined by the
number of fibres arising fi'om a single seed. The total number of fibres
may also be derived fn>m the runnber of fibres arising from a unit ana
of seed coat surface — a number tt> wliieh the term "density" will be
applied. Calculated in terms of "density " tiie number of fibres will be
seen to be goveiiied by the area of seed coat surface which, in its turn,
is I'xprt'ssible in terms of volume. Tiiis consideration is of advantage
inasmuch as it indicates tiiat the weigiits of both the seed and fibre are
directly affected by the same character — the volume of the seed. Whde,
however, the weight of seed will vaiy as the cube of the seed radius, the
weight of lint will, if the " density " remains constant, vary as the surface
area, which varies as the s(|uare of the seed radius. Hence, other factors
being constant, the ginning percent will increase as the volume of the
seed diminishes. The calculations in terms of " density " are, however,
open to objection inasmuch as the number of fibres is probably deter-
mined eail}- in the course of development of the ovule while the volume

H. M. Leake 43

of the seed, and hence the surface area and " density," will, in part at
any rate, be determined later by the nutrition supplied to the ovule
throughout development. It seems, therefore, advisable to use the
number of fibres arising from a single seed in preference to the figure
obtained for the density for the fibres.

From such a priori considerations, there is reason to suppose that
the ginning percent actually recorded will be the resultant of at least
the four characters :

(1) Volume of seed,

(2) Specific gravity of seed,

(3) No. of fibres arising from a single seed
which may be simple characters, and

(4) Weight of the individual fibres, obviously a complex character.
A further consideration will show that the problem is probably even

more complex. The seed and lint are developed in a closed cell of the
fruit, each cell of which contains a number of seeds, frequently eight.
According to the conditions of environment, the nature of the tissue,
and, consequently, the space in which the seed must develop, will vary.
It is probable, therefore, that, during the course of development an
unknown, but appreciable, effect is produced bj' the mutual pressure
thus brought into play. Conclusive evidence of the exact extent of such
nmtual pressure is not so far available. It is clear, however, that the
effect will not directly influence the ginning percent but that such effect
as it may produce will be indirect and through one or other of the four
characters already noted above. The importance of a recognition of this
nmtual pressure lies in the fact that the actual values obtained for the.se
characters may not represent the true potential capacity of the jjlant.
This has already been noticed by Balls'. The present discussion, which
concerns the factors directly controlling the ginning j^ercent, is not
affected therebj', and the problem as it now stands may be defined as
the determination of the degree to which each severally of the four
characters given above influences the ginning percent and of the extent
to which they, in combination, account for the range of variation recorded
in that character.

Methods. In a preliminaiy note of this nature it is not possible to
give in detail the precautions it has been found necessary to adopt.
The main outlines of the method only can be indicated. The unit used
as a sample is the seed-cotton derived from a whole and not diseased

' Balls, W. L., The iottoii plant in F.yijpt, p. 170.

44 Ginnhif/ Percent of Indian Cottons

boll. The sample is gathered after the capsule has expanded thoroughly
and when the lint and seed are thoroughly drj'. From this sample fibres
to the number of about 2000 are accurately counted and weighed. The
remaining lint is removed from the seed by a small hand gin and the
weight of lint and seed recorded as is also the number of seeds. The
volume and specific gravity of the seed are then determined by displace-
ment in water. The data thus obtained give

(1) weight of a known number of fibres,

(2) weight of total fibre,

(3) weight of seed,

(4) number of seeds,

(5) volume of seed,

from which the figures required can be derived by direct calculation.

The method is not ideal as, apart from the precautions necessary in
the process of sampling, a single complete determination, with subsequent
calculation, occupies at least two hours — a point of considerable import-
ance where the value of the results is to a large extent determined by
the number of the observations.

During the course of the investigation it soon became apparent that
the specific gravity of the seed, even within the limits of a single sample,
was subject to marked fluctuation. Evidence, as far as it was obtain-
able, indicated, however, that such fluctuation depends on the conditions
which prevail during the process of ripening. Any inherent dift'erence
in specific gravity due to the nature of the plant is small in comparison
with such observed fluctuation. It has been considered advisable,
therefore, to eliminate this character by reducing the specific gravity
in all cases to an uniform figure of I'lO, such a correction involves a
coiTesj)onding correction of the observed giiming percent. There thus
remain three characters, volume of seed, number of fibres arising from a
single .seed and weight of the individual fibres, which, from the given
a priori considerations, there is reason to suppose might influence the
ginning percent. The object of the present work is to determine how
far fluctuations in the ginning percent can be accounted foi- by these
characters only and whether a search for other characters must be
undertaken.

The units used for the expression of these values are
volume of seed ... cubic millimetres.

No. of fibres per seed ... in thousands,
fibre weight ... weiaht «if 1000 fibres in milliorams.

H. M. Leake 45

The material on which the preliminary determinations, here dealt
with, have been made consists of 232 samples derived from a wide series
of plants of the Asiatic series cottons, and covers, as far as possible, the
full range of variation found. Full details cannot be given here, but a
few typical instances illustrating the range may be recorded.

Seed
/olume

Number of fibres
per seed

Fibre
weight

Oin percent

Remarlis

67

1-2

8-1

11

A form from China

66

2-9

7-4

23

An F^ plant

45

3-3

6-6

30

G. arboreum

67

3-3

10-5

31

A form from China

36

5-2

3-7

34

G. ner/lectum, yellow fjowered

62

7-6

5-9

40

G. neglectum, white flowered

42

6-3

.5-9

44

5) n

These few illustrations are sufficient to indicate the wide degree of
variation found in the characters under consideration, and it is note-
worthy that there appears to be no direct relation between the ginning
percent and any of the remaining characters, even in the selected series
here given. That this must be so is again illustrated by abstract
consideration. I am indebted to Mr Udny Yule for the following
formula which indicates the relation between the four characters
concerned :

number of fibres per seed x wt. of 1000 fibres

where k = r r j •

volume 01 one seed

From this it is clear that the ginning percent cannot be directly
measured from any single one of the characters here dealt with. It is
necessary, therefore, to proceed further and calculate the coefficients of
correlation.

For this purpose the characters may be numbered as follows :

(1) Ginning percent,

(2) Number of fibres per seed,

(3) Wt. of 1000 fibres,

(4) Volume of seed.

The correlations between these characters are found to be

ri2+-7933

E±-0164

J-13+-0530

£±•0442

ru - -2208

E ± 0420

7-23 --2285

£±•0421

r24+-0966

£±•0439

r34+-5147

£±•0326

46 Gmiihif/ Percent of Indian Cottons

From these tiguivs the . j)artial correlation eucfHcieiits may be
ealciilated. These have been determined as follows':

r,,.4+-8392 r,2.34+-!l7W)

r,3.4+-1992 (-,3.24+ -9198

r23.,--3260 ru 23 -■9282

(■,2.3+ -8285 )-23.i4- -925.5

/•U-3--2898 r24.,3+-92fif.

rs4.3+-2566 r3,.,..+ -9316

n3.2+-3952
In. 2- -^909
1-34.2+ -5540
'•23.1 -•■4449
(-24.1 + -4577
r34.i + -5406

From a consideiaticjii of these coefficients it is clear that the tonr
characters concerned form a closely interrelated gi'onp in which variation
in any one character is very fully accounted for by variation in one or
other of the other three. Further, of the three characters by which the
ginning percent may be affected, one only, namely the number of fibres
per seed, has any marked effect on the value of the ginning percent.

Certain other conclusions may be drawn from the above figures of
which one or two may be mentioned here. In the first place the high
negative correlation (r = — '9282) between the ginning percent and the
volume of the seed indicates the validity of the conclusion, based above
(p. 42) on a priori considerations, that the ginning percent, other
factors being constant, will increase as the volume diminishes. Secondly,
the high negative correlation between the number of fibres per seed and
the weight of 1000 fibres (r = — •9255) seems to indicate that the area
in which the fibre can develop is limited as a result of which any
increase in the number must be accompanied by a diminution in the
space occupied by each individual fibre. This conclusion has already
been foreshadowed above.

Lastly, while variation in the number of fibres per seed will produce
a marked direct effect on the value of the ginning percent, variation in
either the fibre weight and the volume of the seed can produce but a
small effect in that the correlation between these and the iiund^er of
fibres per seed is low (»-23= — -2285 and ?-,4 = — •0966).

The main and all-important conclusion, however, to be drawn from
these results lies in the fact that there is here definite proof that the
ginning percent is a complex character, the variation found in which can
be almost completely accounted for by the variation found in the three

' Cf. Yule, An introduction to the Theory of Statistics. 2nd edition, p. 241 et seq.

H. M. Leake 47

characters, number of fibres per seed, weight of the individual fibres
and volume of seed. It follows, therefore, that the determination of
the ultimate causes of variation in the ginning percent cannot be made
directly. Rather, those must be sought indirectly through their effect
on the three characters under consideration and especially on the
number of fibres per seed.

The conclusion just reached opens up a wide field of investigation.
Before practical use can be made of the above conclusions in plant
breeding it is necessary to determine the true value which can be
assigned to any given plant for these characters. In all observations
involving multiple characters a considerable fluctuating variability is
found in different samples from the same individual, and it is necessary
to determine the extent of such fluctuations. The somewhat tedious
nature of the determinations militates against any rapid conclusion
being arrived at and, though considerable progress has been already
made, a full exposition of the results must await further experiment.
Sufficient information has, however, been obtained to show that
such individual variations are of a magnitude readily distinguishable
from the differences found between different races. A single instance,
based on determinati<in of the number of fibres per seed, must here
suffice.

Average

number of

fibres per seed

Maximum

range
of error

A single plant culture of Type 9^

4561

762

A field crop of Type 9

6279

2761

There is thus good reason for believing that further investigation
will make it possible to elucidate such cases as that given above where
two plants, both having a ginning percent of 25 — 26, when crossed gave
rise to offspring of which the ginning percent varied from 36 — 18.
There is also hope that the ultimate physiological causes of fluctuation
in the ginning percent may be traced.

In conclusion the writer desires to express his great indebtedness
to Mr G. Udny Yule and to Dr G. T. Walker, F.R.S., of the Indian
Meteorological Dept., for their kindly help, also to the Authorities of
the Imperial College of Science and Technology and especially to
Prof. J. B. Farmer, F.R.S., for the facilities given him at that College
for carrying out these investigations during a period of leave from India,
and lastly to his sisters, too numerous to mention individually, for their
kindly help in checking the somewhat lengthy calculations.

' For an account of the types see Leake, Journal of Genetics, Vol. i. No. 3 (1911),
pp. 209—211.

IMMUNITY TO FUNGOUS DISEASES AS A
PHYSIOLOGICAL TEST IN GENETICS AND
SYSTEMATICS, EXEMPLIFIED IN CEREALS.

By N. I. VAVILOV,
Agricultural Higher School, Petrovsko'ie-Rasumovsko'ie, Moscow.

CONTENTS.

PAGE

Specialization of Parasitic Fungi .......... 50

Causes of Immunity and Susceptibility in Plants 50

General Idea of the Connection between Immunity of Organisms and their

Genetics .............. 51

Examples in Literature of the use of Fungal Reactions as physiological tests to

aid Systematic and Genetic Enquiries ........ 51

Application of this method for the purposes of the Systematic Study of Cereals

(1) Recognition of Races 52

(2) Example of " Persian Wheat " 53

(3) Example of Hordamii distichiun var. nudidejiciens Kcke. ... 54
Application of Fungal Tests to Cereals for the purpose of elucidating the Genetic

Relations of same ............ 55

((() Characteristics of the eight species of Wheat in relation to Rust and

Mildew 55

(6) Characteristics of Oats in relation to

(1) Crown Rust 57

(2) Black Eust 58

(3) A note, concerning Ergot of Wheat 58

(c) Fungal Reactions of Species of Wheat and Oats, and their Genetic

Relationships 59

(1) Tr. monococcum 60

(2) Tr. vuli/arc and Tr. compaclum 60

(3) Tr. durmii, Tr. pulonicum and Tr. turgidum .... 60

(4) Tr. dicoccum 60

(5) Avena saliva, A. fatua, A. Ludoviciana Dur. and A. sterilis L. 61

(6) A. strigosa and A. brevis 61

" Bridging Species " as an objection to the application of Fungal Reactions for

Genetic Purposes ............ 62

Conclusion 63

Literature 64

Journ. of Gen. iv 1

50 Immimiti/ in Cereals

One (if the t'uiidanicntal pheiioincna in the parasitism of fungi on
plants, playing a decisive 7-ule in the selection of immune sorts, is the
specialization of parasites. The majority of pai'asitic fungi are, by their
nature, sharply limited in the choice of their hosts, and are attached to
definite genera and species of plants. In many cases they are limited
to one plant genus. In others, especially in the group of rusts and
mildews, there are cases in which the fungus is limited to a few, or even
to one host-species only.

In rusts, mildews and a few other families of fungi, the differentiation
went so far that the same morphological species are not seldom divided
into many independent physiological races, so-called " biologic forms,"
which are attached to different host-jjlants. In some instances the
various stages of fructification of the same fungus may be differently
specialized'.

The study of the causes of the imniunity and susceptibility of plants
hitherto has not made much headway ; the phenotnena of parasitism
being too comj)lex, and the drawing up of general statements a matter
for the future. But still it has been shown, by the works of Eriksson,
as well as by Marshall and his school (17), that immunity depends,
not on the anatomical peculiarities of plants, but on the properties of
their protoplasmic cell-contents. Salmon and Miss Gibson have also
establisheil that positive chemotactic attraction of the germ tubes of
fungi by the juice of the host-cells, is not sufficient to produce the
normal growth of fungi on the plant. In fact it is now clear to us
that immunity depends on very complicated physiological inter-relations
between the protoplasm of host-cells and fungus, and that the external
differences in regard to immunity to fungal diseases, which are per-
ceptible in various races of plants, are an indication of internal hereditary
differences in the constitution of their plasma.

Starting from the nature of immunity, as we undcrst;ind it at the
present day, and from the fact of the specialization of parasites—
the connection between the phenomena of immunity and genetics
becomes evident. It is obvious that fungi, and, in particular, narrowly
specialized ones, may be used in some cases as a physiological test (or,

' A very interestiiiR case of this was recently reported by J. Norton for the autoecious
rust of asparagus, Puccinia asparagii D.C. lu regard to tlie " uredo '' or summer stage,
some immune sorts of asparagus have been found, but these resistant plants are all
susceptible to the same fungus in the "aeoidio" stage. {Methods used in breeding
Asparnpus for Rust Resistance, by .J. B. Norton. Bureau of Plant Industry, Washington.
Bui. No. 263, 1913, pp. 23—24.)

N. I. Vavilov 51

strictly speaking, reagent) for the recognition of the species and races
in systematic and genetic studies of plants.

The general idea of a connection between immunity ;ind genetics is
certainly very conspicuous, and there are many data to prove it. It
is enough to mention the well-known fact that nearly allied species
of animals are very often susceptible to the same diseases ; nearly
related genera of plants very often suffer from the same insects. Several
genera of parasitic fungi are exclusively connected with definite families
of plants, as for example Phragmidium with the family Rosaceae.

The general indications of the possibility of applying fungal reactions
of plants in ascertaining their affinity, may be found in mycological
literature. But, notwithstanding the certainty of a relation between
immunity and genetics, very few cases are known of the actual use of
fungi or other parasites as tests in genetic and systematic work. There
are probably two reasons which explain the neglecting of this method :
firstly, because in the majority of cases, fungi are not sufficiently
specialized to be employed for the delicate differentiation of nearly
related plants, and secondly, on account of the usual divergence of the
work of pathologists, genetists and systematists.

As to examples in literature of the use of parasites as physiological
tests, I can cite very few. First, Prof Klcbahn in his monograph on
rusts (7), pp. 140 — 141, in the paragraph entitled: " Verwendung der
Spezialisierung des Schmarotzers zur Unterscheidung der Arten und
Wirte," mentions a case in which by the aid of narrowly specialized
rust Melampsora ribesii jMrpureae he found a mistake in the denomina-
tion of a willow plant in his garden. A second example occurs in the
work of Eriksson entitled : Ein parasitischer Pilz uls Index der inneren
Natur eines Pflanzenbastards (4). In this work he has shown that the
hybrid of wheat and rye is immune to brown rye-rust Puccinia dispersa
and susceptible to brown wheat-rust P. triticina, which proves that this
plant is nearer to wheat than to rye (16, p. 104). A third case is furnished
by E. M. Vasiliev's observations on the injurious insects of maize in
which he makes an attempt to connect the number of species of these
insects with the origin of this plant (15)'.

' In Darwin's Variations of Animah and Plants under Domestication, second edition,
Vol. II, Chap. XVIII, we find the following footnote about the affinity of Aperea and
guinea-pigB. " I sent to Mr H. Denny, of Leeds, the lice which I collected from the wild
Aperea in La Plata, and he informs me that they belong to a genus distinct from those
found on the guinea-pig. This is important evidence that the Aperea is not the parent of
the guinea-pig; and is worth giving, as some authors erroneously suppose that the guinea-
pig since being domesticated has become sterile when crossed with the Aperea. "

4—2

52 Immunitjj in Cereals

Working both on the imniunity an() systeniatics of cereals, I often
had an opportunity of proving the connection between the relationship
and fungal reactions of these plants. My observations led me to the
conclusion that fungi, as a physiological test, may be very useful in
the genetic and systematic study of this plant gi-oup.

Some of the results obtained have been published (16). Here I
propose to cite several general conclusions from work done, and then
add new data.

To begin with, a few words about the application of fungal tests
for the purposes of the systematics of cereals. At the present time
attention, in the systematic study of these plants, is being directed
towards numerous small constant botanical units, the so-called " races."
The principal justification of this work is a very real need for the
drawing up of a detailed catalogue of cereal races, useful alike to
the worker in genetics and to the practical agriculturist. In the
description of these races not only morphological characters are taken
into account, as is usual in purely systematic studies, but also physio-
logical ones.

The extremely narrow specialization of many fungi found on cereals,
and the well-known fact of the existence of differences in the degree of
susceptibility to diseases amongst various sorts of them, waiTanted
first of all the attempt to use fungal tests for the recognition of the
races.

In fact, by the aid of this method, I was easily able to divide many
varieties' of wheat and oats into races. The important role in the
systematic study of such a character as the degree of immunity is
increased, as practice in its application has shown, by the circumstance
that the physiological individualisation of race is very often accom-
panied by morphological characters which, however, are externally not
very conspicuous. In these cases the peculiarity in behaviour towards
fungi obliges the observer to pay more attention to this or that pre-
viously unsuspected race, and in the end he usually succeeds in finding
in it some other confirmatory differences".

1 We use here the word " variety " in a purely botanical sense. (See F. Koernicke,
Handbuch tier Getn-idc, 1885.) In the group of cereals "varieties" usually are collective
notions, and some of them include many independent morphological and physiological
forms — "races" — the smallest systematic units.

- Parenthetically we must remark that the definition of degree of susceptibility to a
certain disease is not quite simple. In fact, the exactitude of many old works in this
respect is so small that in our days we are obliged to obtain these data anew. More
details about the definition of degree of immunity are given in our paper (16), Chapter i.

N. I. Vavilov 53

With respect to wheat the ajjjjlication of fungal reactions is a
specially grateful method, because in this instance we have at our
disposal many narrowly specialized parasites, in other words many
sensitive physiological reagents, as brown and yellow rusts, mildew,
etc. When one test is not sensitive enough to distinguish our races,
we may have recourse to a second, a third, and so on.

In the above-mentioned paper (16) many examples of such a division
of varieties of oats and wheat into races are given. As a, perhaps,
interesting result, I may mention that many races were found to exist
in some of the less common varieties, as Avena diffusa As. and Gr.
var. hrunnea Kcke., var. cinerea Kcke., var. montana Al., and in several
rare varieties of Triticum vulgare Vill.

In the species Triticum dicoccum, which is represented by many
varieties and races, it was ascertained that there exist two groups of
races : one immune to brown rust, the other susceptible to it. The
genetic significance of this will be dealt with later on. Here we must
observe only, that the resistant forms of Tr. dicoccum, which were in
our collection, are morphologically, in the structure of ears and leaves,
very like several varieties of Tr. durum. The degree of immunity of
these latter to brown rust is also nearly the same as in the resistant
races of Tr. dicoccum.

The most interesting example of the application of this method to
the systematic study of wheat I met with, is the following :

In the investigation of 580 sorts belonging to the species Tr. vulgare,
which in general is very susceptible to mildew {Erysiphe graminis D.C.)
and to different rusts, we found to our surprise a spring race which was
perfectly immune to mildew. Notwithstanding many attempts at arti-
ficial infection in the field, in the greenhouse or under bell-jars, this
race remained quite immune. Not one pustule of mildew was found
on this wheat, whereas other races of Tr. vulgare in these conditions
were severely attacked by the above fungus. This wheat proved also
to be relatively very immune to brown rust (P. triticimi). The seeds
of this wheat were obtained from a German seed merchant under the
name of " Persian Wheat."

Such an extraordinaiy immunity as distinguished this race from
other races of Tr. vulgare and from the majority of I'aces belonging
to other species made me pay exceptional attention to this form, and
the preliminary investigations so far made have revealed many further
peculiarities in it. Although a number of morphological characters
show that it belongs to e(jnnnon wheat, namely to Tr. vulgare var.

54 Immunity in Cereah

fuliginosmn M? — a variety with black bearded hairy (;ars and led
grains — yet this form is distinguished by uiany other characters from
all our races of Tr. vulgare, which are not inehided in the chissification
of Koemicke.

The straw of this wheat is relatively full of pith. TTsually, the varieties
of Tr. vulgare have a hollow straw. The stem-knots of this wheat are
visibly hairy; generally they are smooth in the species Tr. vulgare-
The rachis of " Persian Wheat " is only half as broad as in common
wheats. Ordinarily in wheat, only flowering glumes possess an awn.
In this " Persian Wheat," the empty glumes are also awned'. Besides
these, there are other small inorpholugical differences in the structure
of ear and leaf.

By crossing this wheat with other varieties of Tr. vulgare, it was
observed that the percentage of successful results was \ er^' small. The
same fact was repeated the next year. Usually in the same conditions
the percentage of success in our crossings of different varieties of Tr.
vulgare was very high. In the Ft hybrids ($ "Persian Wheat" x (/ Tr.
vulgare var. lutescens Kcke.) about 70 ^ of the spikelets were sterile.

Finally in some of our crossings of susceptible races of wheat with
immune ones, the susceptibility to mildew was clearly dominant, and
F^ hybrids were severely attacked by this fungus, as was seen in the
experiments of Prof Biffen with barley. But in the case of crossing
"Persian Wheat" with a very susceptible race (Tr. vulgare var. lutescens
Kcke.), only after many efforts did we succeed in infecting slightly the
F^ hj'brids, when in the same conditions the susceptible parent was
severely attacked. In other words immunity in this instance is not
" recessive."

\yithout dwelling further on this case, we must remark only that
all that has just been said about this wheat allows a systematist to
separate it from other varieties of Tr. vulgare, and makes it of special
interest for the genetist.

An instance I came across in the investigation of barley is analogous
to that described by Prof. Klebahn with willows. In our collection we
had some samples of naked two-rowed barley from different parts of
Ru-ssia. In the classification of Koernicke (1885), which we made use

' Tlie name Tr. i-ulgare var. fuliginosum Al. is a collective name. We know quite
ordinary susceptible wheats of the species Tr. vulgare which have black, hairy, bearded
ears with red grains, like "Persian Wheat," and which must be placed in Koeruicke's
classification under the name " var. /H/((/(«oj>»»t. "

-' This character is observable also in a few races of Tr. vuhjarv, belonging to different
varieties, which are cultivated in Asiatic Russia and Persia.

N. I. Vavilov 55

of for the identification of barleys, all naked two-rowed barleys are
represented by one variety — Hordeum distichuvi var. nudum L. To
this variety we had referred, after identifying, all our naked two-rowed
sorts of barley. But from observation of these barleys during two years,
it was noticed that one of these parts (a pure line) was noticeably less
susceptible to Puccinia simplex Eriks. than others, although growing
side by side. This circumstance obliged me to pay more attention to
the form in question, and in the result it was found that we had a very
rare variety, which was wanting in the old classification of Koernieke.
It is distinguished from var. nudum L. by weak development of the
lateral spikelets (as in var. deficiens Steud.), and it was described only
in the posthumous article of Koernieke (8), published in 1908, under
the name of Hordeum distichum var. midideficiens Kcke. We i-eceived
this variety from Caucasus (Daghestan).

I now append some examples of the connection of the fungal reaction
of cereals with their genetics.

Ghiracteristics of the Eight Species of Wheat in relation to Rust

and Mildew.

After the work of Prof Biffen and Nilsson-Ehle, which proved that
immunity and susceptibility to fungal diseases is subject to the Men-
delian rules of inheritance, it would seem very natural to suppose that
the distribution of these characters amongst hundreds of varieties and
races of cereals is (piite accidental and without any definite order, as
immunity and susceptibility may be combined by the aid of crossing
with any group of morphological characters. Efspecially it would be
natural to suppose it to be so in such a group as wheats, seven sjjecies
of which {T. vulgare, T. compactum, T. durum, T. polonicum, T. tiirgidum,
T. Spelta and T. dicoccum) have been proved to be fertile by crossing'.

In reality, it is far from being so.

After investigation of about 800 sorts (represented by pure lines) of
spring and winter wheat, collected from diffei'ent parts of Europe and
Asia, with regard to fungi prevalent in European Russia {Puccinia
triticina Eriks. and Erysiphe graminis D.C.), and after classifying the
sorts and tabulating these data, we came to the conclusion that in
general each of the eight species of wheat, including dozens of varieties
and races, has a definite characteristic behaviour in relation to fungi
(16, pp. 29—54, 94—102).

' See works of Vilmoiin, Beijerinck, Eimpau, Tschermak, Biffen and others.

56

Inuminitf/ hi Cereah

For example, all the cultivated vaiieties of T. durum and T. turgidum
are relatively immune to brown rust. All known wikl and cultivated
varieties of T. luonococcum are perfectly immune to brown rust.

Many scattered data, which were found in old and recent literature,
referring to the same or other varieties, prove the correctness of this
conclusion (the literature is given in the above-mentioned paper 16,
pp. 94 — 99, 5 — 6) and allow us even to apply, in some degree, this
generalisation to the relation of the species of wheat to other fungi,
as yellow ru.st P. glumaruin. For instance, all varieties of T. mono-
coccuni, in accordance with published data, are perfectly immune to
yellow rust. The different varieties of I', durum and turgidum are
relatively immune to the same rust.

In general, the characteristics of the eight species of wheat in
relation to the fungi by which they are attacked in Russia are as
follows :

resistant

Tr. durum Desf
Tr. polonicum L.
Tr. turgidum L.

In relation to Puccinia triticina Eriks.'

xusceptible

Tr. vulgare Vill. (thei-e are a few

immune races)
Tr. compactum Host.
Tr. Spelta L.

perfectly immune

Tr. manococcuiii L.
Tr. dicoccum Schr. has both susceptible and inumme races.
In relation to Ery.nphe gra minis D.C.

tfuseeptihle resistant

Tr. vulgare Vill. (with the exception Tr. durvm Desf.

of a few races) Tr. polonicum L.

Tr. compactum. Host." Tr. turgidum L.

Tr. Spelta (a little less than the T?: monococcum L.

preceding ones).

Tr. dicoccum Schr. has both susceptible and iiiuuuue races^

• lu the paper (16) are given the coloured plate.s, illustrating the differences iu suscepti-
bility of wheats and oats to brown, black and crown rusts.

- Only one race, belonging to the variety Tr. compacliiiii var. creliciim Mazz. proved to
be relatively resistant to mildew. (The same was observed in America by Prof. Keed.)

'■> These characteristics of species are based on observations in fields under different
conditions of manure, soil and climate. In greenhouses brown and yellow rusts do not
develop to any considerable extent even by artificial infection ; on the contrary, the

N. I. Vavilov 57

Only in Tr. vulgare and partly in Tr. coriipactum there are a few
relatively immune races — exceptions to the general characteristic of
these species, as susceptible to brown rust and mildew. One of the
extreme exceptions is the above-mentioned " Persian Wheat " ; several
of the other more or less immune races, without any doubt, represent
products of artificial crossing in recent times (16, p. 96).

Also, briefly speaking, in the group of wheats we meet with a case
of specific peculiarities of whole species in their fungal reactions, not-
withstanding the great polymorphism of these species.

The genetic significance of these data I shall shortly touch upon.
The practical importance of this generalization for the selection of
immune sorts is evident, for it simplifies considerably the work of the
plant-breeder.

Characteristics of the Species of Oats in relation to Rusts.

As in Russia, so in England and other countries, oats are attacked
very severely by two species of rust : crown or leaf rust P. coronifera
Kleb., and black or stem rust P. grariiinis Pers.

Observations in Moscow showed that the majority of cultivated and
wild oats are very suscej)tible to crown rust.

Of 323 sorts of Avena sativa L. (.4. diffusa Aschr. and Gr., and
A. orientalis Schreb.) examined, 297 belonging to the majority of
known botanical varieties of cultivated oats (8) proved to be very
susceptible ; 21 less susceptible, and 5 races (belonging to the varieties
var. cinerea Kcke., var. brunnea Kcke., and var. grisea Kcke.) proved to
be relatively very immune to crown rust. The great majority of these

conditions of greenhouses are very favourable to mildew of cereals ; the fungus in
the conidial stage lives, for example, in greenhouses much longer than in the open
air, and in general the plants are always more attacked by mildew in greenhouses than
in fields. And even more or less resistant races of wheat, for instance different repre-
sentatives of Tr. durum, polonicum, maybe severely attacked in the greenhouse, as also
under the bell-jar, by Erysiphe graminis. Immune races do not " lose" their immunity
in greenhouses. The difference in susceptibility may be observed during the first days of
infection, but the fungus develops better under these conditions. The most important
fact is that even under these conditions such races as "Persian Wheat" or several races
of Tr. dicoccum remain uninfected.

By what is said above is removed the controversy relating to the characteristics of
Tr. durum and Tr. polonicum in our work and that of Prof. Reed in America (Pliyto-
pathology. Vol. ii, No. 2, 1912), who defined the degree of susceptibility of 78 sorts
of wheat by the aid of artificial infection of seedlings under bell-jars. Tlie data of the
characteristics of other species of wheat, in Russia and America, in general coincide.

58 Immunity in Cereals

last 26 races belong to vaiieties with black and grey grains (Howering
glumes), and in general they are morphologically different from sus-
ceptible races (IG, pp. 15—18, 94—95).

The various examined races of wild oats, belonging to the species
A. fdtuu Ij., A. Ludoviciana Dur., and ^1. sterilis L., all pioved to be
very susceptible to ciown rust.

Aveiui brevis Roth., A. striyosa Schreb., and A. inula L. var. biu-
ristata A.seiir. and Or. are relatively immune to this rust.

A different result was obtained with black rust. Of S50 examined
sorts of cultivated and wild oats, belonging to nearly all known botanical
varieties, only two races of the species A. sativa L. proved to be
relatively immune, (^ne of them (more resistant) belongs to the var.
bruiinea Kcke., the other (less resistant) to the var. montana AL, two
varieties with dark flowering glumes, and both these races are morpho-
logically very different from the other susceptible races of the same
varieties ; for instance, they are very low plants and are characterized
by very thin straw; practically both are of small value. All other
cultivated and iuitd varieties belonging to six species are badly attacked
by P. graiiiinis.

In other words, as a result of these observations, we come to a
simple statistical conclusion as to the very slight probability of plant-
breeders' finding oats resistant to black rust.

This conclusion will be rpiite logical if we remember that black
rust of oats is a very weakly specialized fungus, whieii lives freely not
only on th(^ genus Aveiai, but also on Alopecurus, Milliuni, Bromus,
Lamarckiana, I'htdaris. Koeleria, Festiica and other genera of ^t/'«-
mirieae. For genetists it is (juite natural to conclude that if the
fungus does not distinguish generic differences, there is ver}- little
probability that it will sharply distinguish racial differences in the
species A. sativa L.'

' A similar argument may be applied to the ergot of cereals — Chiviceps purpurea Tul.
The same biologic race of this fungus, according to Stiiger's experiments, lives on rye,
barley, wheat, Aniliojtinllium, HierochUm, Arrlii'inaherum, Daclylin, Foa, Ilri:a anil other
genera of Griimineae. Theoretically, therefore, there is very slight probability for plant-
breeders to find a great difference among races of rye, barley and wheat in their
susceptibility to this fungus. The great difference between rye, barley and wheat in
the degree of infection by ergot (the two latter are very rarely attacked by ergot), is
evidently connected with tlie different modes of Howering of these cereals. Rye usually
flowers with opened glumes, wheat and barley with closed glumes ; and the closed mode
of flowering prevents the two latter from being infected by ergot. Eventually all the

N. I. Vavilov 59

Fungal Reactions of Species of Wheat and their Genetic
Relationships.

Now turning again to the general characteristics of the species of
wheat and oats in relation to narrowly specialized fungi, we shall see
that they have not only significance for plant-breeders, but deserve
serious attention on the part of students of genetics. As is known,
the genetic relations of cereals are far from being solved. Every new
criterion for the understanding of this problem is useful and valuable.
It is especially so because the usual criterion of degree of affinity —
sterility or vice versa — fertility of hybrids cannot always be used in
the group of cereals. For instance, the seven species of wheat are
so nearly allied that they give fertile hybrids. To understand the
genealogy of this group, botanically restricted but nevertheless repre-
sented by an immense number of independent forms, we must employ
finer methods.

On looking into the characteristics of eight species of wheat in relation
to rusts and mildew, we cannot help being struck by their complete
agreement with several genetic conceptions which are more or less
established concerning their relationship.

eight species of wheat, according to our observations in Russia, may be slightly attacked
by ergot, especially when wheats are cultivated side by side with rye.

In his second paper on " Studies in the Inheritance of Disease-resistance," Journ. of
Agr. Sc, Vol. iv, Part 4, 1912, Prof. Biffen communicates a curious fact of the occurrence
in the F„ hybrids of Eivet (Tr. tur<i'uium L.) with several varieties of Tr. rulyare of some
plants which were attacked by ergot, although the parent forms had never been seen
to be attacked by this fungus. Prof. Bift'en explains this fact, as a result of combination
of two Mendelian factors of susceptibility to ergot, which are separated in their parents,
and in separate form cannot produce the susceptibility of wheat to ergot.

The apparent contradiction of this case of distinct difference in susceptibility to ergot
of wheat plants to the above-mentioned general statement, however, is easily removed by
a simpler and more probable interpretation of this fact, than that given by Prof. Biffen.

Already in 1891 Prof. Rimpau, in his Kreusungsprodukte landwirtschaftlicJier Kultiir-
pflanzen, pp. 11 — 12, had noticed the fact that in the same crossing of Rivet and Tr.
vulgare there appear in F„ hybrids some slerile ■plants. The sterile plants of cereals as
is known flower usually with open glumes, remain many days in this state, and commonly
are badly attacked by ergot. (See E. Tschermak, "Die Bliih- und Fruchtbarkeitsverhalt-
nisse bei Roggen und Gerste und das Auftreten von Mutterkorn," Fiihlings landwlrtsclmft.
Zeitung, Lx. 1906.) The sterile plants of F^ of the hybrid of wheat and rye, for example,
are severely ergotized.

Evidently the same fact of appearance of sterile plants was observed in the experi-
ments of Prof. Biffen at Cambridge, and, as might be suppo.sed, these sterile plants were
attacked by ergot.

00 lianmnitji hi Cereals

(a) Tr. iHOiiococcunt L. is iiiiaiiiuiously separated by gunetists as
an independent species fniiii all tlie other seven, principally on acconnt
of the sterility of its hybrids with the other seven species of wheat,
which has been proved by many investigators [Vilniorin, Beijerinck,
Koernicke, Tscherniak](18), whereas all these latter (2V. vid<jare, com-
pactum, dicoccttm, tmr/iduni, durum, pulonicain, and Spelta) in crossing
give more or less fertile hybrids'. The wild progenitors of onr cnltivated
varieties of Tr. monococcum have been known for a very long time,
whereas the wild prototypes of other species were found only a few
years ago.

The genetic individualisation of Tr. monococciuu is confirmed too by
its fungal reactions. All its known wild and cidtivated varieties are
perfectly inniiune to brown and yellow lusts, and in this respect occupy
a separate jjlace among other species of wheat. In literature we find
also indications that they are equally immune to stinking smut, TiUetia
tritici{6).

(b) Tr. compactum Host. — dwarf wheats, according to modern views
are so nearly allied to common wheats, Tr. vidgare Vill., that many
authors unite them into one species. As we see in the table given
above, their fungal reactions on mildew and brown rust are the same.

(c) Tr. puloiiicvm L. and Tr. turgiduin L. by systematists and
genetists (Koernicke, Schulz, Beijerinck and others) are considered as
species nearly allied to Tr. durum Desf. " Bei einzelnen Formen"(of
Tr. turgiduin) — says Schulz — " kann man zweifeln, ob man sie zu Tr.
turgiduin oder Tr. duruiu zurechnen soil" (10, p. 150). " Tr. polonicuin"
— says Beijerinck — " ist ohne Zweifel nur eine halbmonstrose Abart von
Tr. durum " (3). " Es giebt unter Tr. durum " — says F. Koernicke —
" Sorten, deren Korner in der Liinge, Glasigkeit und der hellen Farbe
genau denen von Tr. polonicum gleichen " (8, p. 897). These three
species are alike not onl}' in the structure of their ears, but also in their
vegetative organs.

The characteristics of these species in their fungal reactions to
P. triticiua, P. glumaruiii and Erysrphe graminis are identical.

(d) Tr. dicoccuiii, Schr. — Emmer — according to the current view,
is a polymoi'phic progenitor species from which the su.sceptible and

' Oulj quite recently M. Blaringhem reported in C. R. ile I'Acad. ties Scienc, 1914,
T. 1-58, No. 5, that he suceeedetl in obtaining a few fertile livbrids ? Tr. monococcum L.
var. vulgure Kcke. x J Tr. iliiriim var. Macaroni, as a result of many crossings of these
species. Many of his other crossings of Tr. mDnornmim with different species, proved to
be unsuccessful or sterile.

N. I. Vavilov 61

immune species as Tr. vulgare. Tr. durum and others (except Tr.
monococcum) are descended. This view, as is known, has been con-
firmed in our days by the finding of numerous forms of wild Tr.
dicoccoides in Palestine and Syria by A. Aaronsohn, and in Persia by
Strauss. In accordance with this view we find in the species Tr.
dicoccum, races both immune and susceptible to mildew and brown rust.
The susceptibility to brown wheat rust and mildew of some races of
wild Tr. dicoccoides, which were kindly sent to me by Mr A. Aaronsohn
and were examined in Moscow in their relation to fungi, once more con-
firmed their near relationship to cultivated wheats. Prof Tschermak
and A. Aaronsohn proved also that Tr. dicoccoides gives fertile hybrids
with cultivated wheats (14).

Funded Reactions of Species of Oats and their Genetic
Relationships.

The same parallelism of fungal reacti(jns and the genetic relations
of plants is observable in oats. According to the present views which
are based on systematic study and experiments in the crossing of wild
with cultivated forms, oats have a polyphyletic origin. According to
recent work by Dr Trabut in Algeria (13), Dr Thellung in Switzerland
(12) and A. F. Malzev in Russia, we regard A. fatua L., A. sterilis L.
and A. Ludoviciana Dur. as the ancestors of our cultivated forms
A. sativa L. (A. diffusa Asch. and Gr., and A. orientalis Schreb.)^

As was said before, the representatives of all these species examined
in Moscow proved to be equally susceptible to the narrowly specialized
crown rust P. coronifera, like the majority of cultivated oats.

A. strigosa Schreb. and A. hrevis Roth., two rarely cultivated species,
which are morphologically much alike, and which by our crossing in
Moscow proved to give fertile hybrids^ are relatively immune to crown
rust. In this respect, they are distinguished from the above-mentioned
wild and cultivated oats.

Many attempts at crossing of A. strigosa (two varieties) with culti-
vated A. sativa (A. diffusa Asch. and Gr.) repeated during two years in
the Moscow Agricultural Institute proved to be unsuccessful, whereas
under the same conditions the crossing of A. fatua. and A. sativa. was

1 The origin of naked cultivated oats, represented by very different morphological
forms hitherto, is far from being clear.

2 This crossing was done after their similar reaction on fungi was known to us, and
in this case the similarity in fungal reaction suggested the possibility of crossing these
two species.

62 I imiiKiiitii ill Cereals

successful and the hybrids wore fertih'. This fact confirnis the peculiar
genetic place of A. strigi>.sii aumtigst other cultivated oats, which was
suggested by its fungal reaction.

Finally, the isolated genetic position of A. strujusti is proved by
the fact, which we find recorded ( I I ), that it is also iniuume to smut
UstiUnjo tivemte, whereas the cultivated forms of ^1. sativa are usually
severely attacked by this fungus'.

Some other examples might be given illustrating how, by the aid
of genetic knowledge, the differences of various cereals in their be-
haviour to fungi become clearer; and vice versa how fungal reaction
helps us to understand the genetic relation of plant forms. But the
complete enumeration of all these examples would be out of place in
this paper.

One objection may be raised against the broad application of fungal
reaction for genetic purposes.

This is the phenomenon of so-called " bridging species" — cases in
which the biologic form of a fungus, after living on certain of its host-
species ("bridges"), becomes capable of infecting a species, which it
cannot infect after living on its other host-species. Pole Evans also
showed with black rust of wheat 1\ gniminis, that very susceptible
F^ hybrids of immune and susceptible varieties may serve as a "bridge"
between susceptible and immune sorts.

But against this, in the first place, there are only very few cases
known of existence of " bridging species-." In the case of fungi of
cereals, they are found oidy in Pttccinia gni minis forma sp. tntici and
not in P. glumurum, r. triticimi, P. simplex {2, 5). Furthermore, we
must not forget that the biologic forms of P. r/raiidnis an- relatively
weakly specialized fungi; for example, /br;/i(( sp. tntici, as has been
shown in different countries, can infect not only wheat but, more or
less, barley also^.

' Morphologically A. slrhjusa is very like A. burbata Pott.; and Dr Trabut and
ThellunR account the latter as the progenitor form of the former species. It would be
very interesting, therefore, to know the fungal reaction of A. biirbatii.

- "Bridging species" are found in P. Si/mphyti Bromvruiii ¥. Miill. (M. Ward and
Freeman), Erysiphe graminis D.C., living on ilrnmiis (E. Salmon), in P. iirdiiiinis forma
sp. tritici (Freeman and Johnson), and Si)liiier(itlii'C(i Hiiniiili on Ah-hciiiilki (Steincr).

■' One of the conclusions to which Freeman and Johnson came after their numerous
experiments with biologic forms of P. yraminis is that "two biologic form.s may inhabit
the same cereal without being identical" (.5, p. 75). This statement, as also the fact,
well-known to mycologists, of the existence in Australia of a different race of P. firaminis
/. tritici, which cannot infect Berberis, and all that is known about the difference in

N. I. Vavilov 63

Secondly, as has already been remarked by Prof. Biffen (2, pp. 428 —
429), two facts speak against the great role of " bridging species " : first,
the fact that immune varieties may be grown dozens of years in close
proximity to susceptible ones, severely attacked by fungi, and still
remain quite resistant ; the second fact is the possibility of obtaining
immune races by crossing.

Finally, it may well be conceded that the exactitude of fungal
reactions must be studied before using them for genetic purposes,
as with reactives in chemistry. With fungi of cereals, this preliminary
work is happily already more advanced than it is with any other group
of fungi'.

In conclusion, we need only remark that the degree of sensitiveness
of fungal reaction, for instance, with cereals up to the present time
is not exceeded by that of the so-called "serum" methods, applied to
plants. It is hardly necessary to add that fungal reaction technically
is much simpler than the " serum " method in its application for the
recognition of individuals.

JIarc/i, 1914.

Note. While this paper was being printed there appeared in the Zeitschrift fur
Pflanzenziichtimg, April 1914, Bd. Ii, Heft 2, a very interesting paper by Dr Zade
entitled : " Serologische Studien an Leguminosen and Gramineen." In his investi-
gation with cereals, using the " serum " reactions as a chemical test for the genetics
of these jilants, Dr Zade, as we understand his tables, conies to quite the same
conclusions concerning the genetic relationshij) of oats and wheats, to which we
came using fungal tests for the same purpose.

So, the tables of experiments on dift'ereut species of Avena show that the
A. fatiM gave almost the same reaction as A. sativa.

A. strigosa,yi\Ac\i according to our investigation is genetically more distant from
A. sativa than A. fatua, gave in the experiments of Dr Zade, when he used the weak
" serum " solution of A. sativa, a much weaker reaction than A. fatua.

specialization of this fungus in different countries, suggest the suspicion that it is possible,
in the same biologic form, more than one race of fungus may exist, and these races differ
more or less in their specialization. And, perhaps, in some instances the same phe-
nomenon of "bridging species" is the result of unconscious selection of different races
of fungi by the aid of different hosts. Certainly this question can be solved only by
means of pure cultures of fungi.

' For the purpose of greater exactness, the conditions of the use of fungal reactions
must be borne in mind too, because, like chemical reactions, they change under differing
conditions ; although in general the role of external conditions (climate, soil, manure,
etc.) in changing immunity is very often too exaggerated in mycological literature (16,
pp. 99—102).

64 Immunity hi Cereals

The similarity in tlio cjiisc of wheats is even more striking. So, Tr. monococcum,
according to Dr Zade's experiments, occupies a separate place. Three species Tr.
durum, Tr. polonicum and Tr. turgidnm, which have the same fungal reactions, had
the same "serum" reaction. The Tr. vidgarc, Tr. com/uic/am and Tr. Spella, both
in relation to fungi and in their " .serum " reaction are very similar.

The similarity of " serum " reaction of Tr. dicoccum with that of Tr. durum,
polonicum and turgidnm. again does not contradict our results ; for probably
Dr Zade used for his experiments a variety of Tr. dicoccum which is immune to
brown rust. It is to be regretted that Dr Zade does not give the nanie.s of the
varieties with which he worked. This makes a complete comparison of results
ditticult.

On account of the great polymoi'phism of tlie species Tr. dicoccttm (and Tr.
dicoceoides) which is very important for the construction of the genealogy of wheats
and which was not taken into consideration by Dr Zade, we cannot agree completely
with the concluding genealogical table of wheats, given on p. 144. We believe
that the difference in varieties which occurs in species of cereals cannot be neglected
in genetic work.

The striking parallelism of fungal and " serum " reactions once more confirms
the possibility of using both these methods for genetic and systematic purposes.

REFERENCES TO LITERATURE.

1. BiFFEN, R. H. "Studies in the inheritance of disease-resistance." Journ. of

Agric. Science, Vol. ll, Part 2, 1907.

2. . "Studies in the inheritance of disease-resi.stancc." II. Journ. of

Agric. Scie7ice, Vol. iv. Part 4, 1912.

3. Beijerinck. " Ueber den VVeizenbastard Tr. monococcv.m x Tr. dicoccum."

1884. A'ederl. Kruidkund. Archif, 2 Ser. p. 200.

4. Eriksson, J. "Ein parasitischer Pilz als Index der inncrcn Natur eines

Pfianzenbastards." Botan. Notizcr, 251—253, 1895.

5. Freeman and Johnson. "The rusts of grains in the United States." Bull.

No. 216. Bureiiu of Plant Industry, U.S. Dep. of Agriculture, 1911.

6. KiRCHNER. " Neue Beobachtungen iiber die Empfanglichkeit verschiedener

Weizensorten fiir die Steinbrandkrankheit." Filhlings btndtnrtsch. Zcitung.
1908.

7. Klebahn. " Die wirtswech.seluden Rostpilzo." 1904.

8. Koernicke, F. " Arten uud Varietiiten des Getreides." Vol. I, 188.5.

9. . " Die Entstehung und das Verhalten neuer Getreidevarietiiten." 1908.

Archiv fiir Biontologic, Bd. II.

10. ScHULZ, A. " Abstammung und Heimat des Weizens." Jahrcsberic/U d. West-

fiilischen Provindal-Verein, 1911, pp. 147 — 152.

11. Swingle. "Report on the loo.se smuts of cereals." Experiment Station,

Manhattan, Kansas, 1889. (Report of the Botanical Department.)

12. Thellung, a. "Ueber die Abstammung, den systematischen Wert und die

Kulturgeschichte der Saathafer-Arten." 1911. Vierteljahrsschrift d. Xaturf.
Ges. Ziirich, Jahrg. 56.

N. I. Vavilov 65

13. Trabut, L. " Observations sur I'origine des avoines cultivees." IV. Con-

ference Internationale de Ge'netique, 1913.

14. TscHERMAK, Er. " Ueber seltene Getreidebastarde." Beitrtige zitr PJlanzen-

zucht, 3 Heft, 1913.

15. Vasiliev, E. M. "The injuriou.s insects of maize in European Russia and

in We.st Europe." South- Russian Agric. Oazette, 1911 (No. 8) and IMS.
Vols. XIII and xv.

16. Vavilov, N. I. "Beitriige zur Frage tiber die ver.schiedene Wider.stauds-

fahigkeit der Getreide gegen parasitische Pilze." (In Russian and German.)
Arbeiten d. Versuchsstation fiir PJkinzenzilchtung am Moskauer Icaidiuirtsch.
Imtitut, I Folge, 1913.

17. . "Der gegenwartige Stand der Frage nach der Immunitat der Getreide

gegen Pilzkrankheiten." 1913, ibid, (in Russian).

18. . "Ueber den Weizen bastard Tr. vulgarey.Tr. monococcum L." (In

Russian and German.) Bull, fiir angcvandte Botanik., 1913, Bn. vi. No. 1,
St Petersburg.

Journ. of Gen, iv

HEREDITARY LEFTHANDEDNESS, WITH A NOTE

ON TWINNING.

(STUDY III.)

By H. E. JORDAN,

University uf Virginia.

This study is based chiefly on data collected by Dr Albert Ernest
Jenks, Professor of Anthropology, University of Minnesota, in the form
of questionnaire blanks and filed at the Eugenics Record Office, Cold
Spring Harbor Long Island. My best thanks are due to Professor Jenks
for his generous contribution, and to Dr Charles B. Davenport for
kindly placing the blanks at iny disposal for examination and record
in the form of charts. The material includes also six pedigrees from
various other sources. Respecting this body of data the primary
objects here sought are to make it more widely accessible by putting
it into more available form, and to analyze it with a view to testing
my earlier conclusions, namely, that lefthandedness is hereditary and
closely follows the behaviour of a Mendelian recessive character. The
questions of the cause and anatomical basis of this trait will not be
touched upon' ; these are similar to those involved in physiologic
"unit characters" generally. Scarcity of data respecting ambidexterity
precludes effective discussion of this character from a hereditary view-
point. The pedigrees include a considerable number of twins ; this
circumstan3e invites a consideration also of hereditary twinning; and
explains the inclusion of pedigree chart Fig. 80.

^ This matter is discussed in my earlier papers.

5—2

68 Hefeditary Lrft handedness

The main body uf data is somuwhat unsatisfactory in respect of the
absence of collateral histories; these are indispensable for complete
studies seeking evidence of Mendelian inheritance; however, the
abbreviated histories are still of the greatest value, and warrant careful
analysis and permanent record.

Three remarkable five-generation discontinuous histories of heredi-
tary lefthandedness are included, charted in Figs. 1 to 3. Here the
absence of collateral histories and record of the total number of
individuals in the several childships is especially regrettable, and
precludes attempts at further analysis.

Four histories of direct transmission are charted in Figs, i to 7.
Chart 6 is noteworthy in that all the affected individuals are males.
Chart 5 suggests recedence of the sinistrality factor, since the Icfthanded
individual of the last generation is the result of a double Icfthanded
mating. However, the three Icfthanded individuals of the preceding
generation contradict this general deduction, since the female parent
was presumably righthanded. Moreover, the fact that the mother had
also 3 nieces, 1 nephew, and 3 great-nieces who were Icfthanded suggests
almost equally cogently that in this family lefthandedness was dominant.
This particular history will be further discussed below in an attempt to
interpret apparent cases of dominant lefthandedness.

The blanks include also eleven, in six of which only an uncle or an
aunt of the Icfthanded individual is known to be similarly affected, and
in five only a great-uncle or great-aunt. Several of these are charted
and discussed below (Figs. 69 — 74).

Forty-eight blanks record lefthandedness in both parent and child.
The histories with more than one affected member in the fraternity are
charted in Figs. 8 to 1(1 This set includes only one instance of double
Icfthanded parentage (Fig. 16). The expected total frequency on
Mendelian assumptions is vitiated by the presence of one " normal."
In the other histories of this set, one of the parents is normal as respects
use of hand. On the assumption that the " normals " are hetcrozygotes
the majority of the charts fulfil Mendelian expectancy for RR x DR
crosses (Figs. 10, 11, 13, 9, and 1-5). Chart Fig. 8 suggests dominance
of the Icfthanded trait, as also to a lessor extent, in view of the limited
childships, charts Figs. 12 and 14. A strict Mendelian interpretation
of this group of charts (Figs. 8 to 16) involves the further assumptions
of degrees of bias to sinisterity and variation in relative hereditary
prepotency. An attempt will be made to .support the legitimacy or
plausibility of these assumptions below.

H. E. Jordan 09

The remaining 39 histories of parent-child "transmission" may be
summarized thus :

father to son ... ... 0

fiither to daughter ... 10
mother to son ... ... 13

mother to daughter ... 7
The numerical variation is small, and of no significance as indicating
prepotency of one or the other parent to transmit the character differen-
tially to either sex.

Adding to these numbers those from cliarts 8 to 16 the same
conclusions become still more obvious, thus :

father to son 9 + 8 = 17

father to daughter ... 10 + .5 = 15

mother to son... ... 18-1-6 = 19

mother to daughter ... 7 -H 5 = 12

The following table summarizes the data respecting fraternities of
four or more individuals:

Numher of leftliamJed and rlghtlianded in fraternities of
four or more individitals. Parent-child histories.

Lett

Right

5

4

3

7

1

4

1

3

3

3

1

3

3

3

2

2

1

5

1

7

1

5

1

6

2

4

1

4

2

7

1

3

1

5

1

4

Totals 31

79

or 1 :

2-55

70 Hereditary Lefthdiidedness

Four fraternities give the expected proportion for a Dli x RR cross ;
several others may be said to approximate expectancy. But only by
assuming that a certain number of the normal consorts were duplex for
dextrality (-D-D), and that the character is occasionally imperfectly
dominant, can the ratio of the totals (1 : 2'55) be made to suggest
Mendelian principles.

Six blanks record lefthandedness appearing in great-grandparent,
parent and child (charts. Figs. 17 to 22). Pedigi-ee chart. Fig. 22, is of
especial interest in that it gives a final generation of two pairs of twins,
all lefthanded. Since both pairs are severally of the same sex, they are
presumably identical ; the fact that identical twins are rarely unlike
with respect of use of hand, suggests very strongly the hereditary
character of this trait. The facts shown in this chart as also in chart
Fig. 17, again suggest dominance of the lefthandedness factor in
certain strains; or "imperfection of dominance" of the righthandedness
factor.

Fourteen histories show transmission from great-grandparent to
child (Figs. 23 to 36). The absence of information concerning collateral
lines, and of complete childships in every instance, forbids deduction
beyond the general statement that lefthandedness in the ancestiy
presages a certain amount of lefthandedness among ofl'spring. The
inference is suggested that the normal parents are heterozygous, in
which event the Mendelian pioportion of 1 lefthanded to 3 righthanded
would be expected. This expectation is practically met in charts 24
and 33. With the exception of charts 35 and 36, the remaining charts
of this group also are in close accord with Mendelian formulae.

Four blanks I'ecord a four-genei-ation history of lefthaniledness with
the direct ancestors of the penultimate generation normal (Figs. 37 to 40).
All show a lefthanded offspring from a double "normal" mating. The
ancestry of the normals concerned, however, suggests their heterozygous
nature ; one in every four would therefore be expected to be lefthanded.
The histories as given are in close accord with this ratio.

Sixteen blanks record lefthandedness in three consecutive genera-
tions (Figs. 41 to 56). Chart, Fig. 41 is interesting in that the direct
ancestors in the two earlier generations were both ambidextrous,
suggesting a specificity of this condition as compared with dexterity
and sinister] ty. In charts, Figs. 44, 50 and 52, the lefthanded condition
is distributed in the fraternities approximately as would be expected in
a DR X RR cross, most probably the actual fact.

H. E. Jordan

71

Hereditary transmission from grandparent to child appears in 44
blanks. The distribution may be summarized thus :

Grandfather to son's son ...

„ „ „ daughter

„ „ daughter's son ...

„ „ „ daughter

Grandmother to son's .son ...

„ „ „ daughter

„ „ daughter's son ...

„ „ „ daughter

The summary seems to show that a grand-daughter rarely inherits
lefthandedness from her mother's father, and a grandson fi-om his
mother's mother ; but the disproportion is due more likely to the
relatively small number of histories as the grosser summaiies show
(second and third columns). In this group again nothing appears
sufficiently striking to indicate prepotency on the part of one or the
other parent (grandparent).

The fnllowing table summarizes the data for this group where the
fiaternities are larger than three individuals :

Grandparent — cli Ud histories

Left

Right

5

7

3

6

4

3

6

4

3

3

6

3

4

4

4

4

Totals

18

69

or

1 :

3-83

72 Herediturji Lefthandedness

On the most probable assumption, namely, that the parents involved
are heterozygous, the expected proportion of 1 to 3 is strikingly and
suggestively met by 1 to 3"83. Moreover, three fraternities contain
lefthanded and righthanded individuals in the exact proportion of 1 to 3 ;
and six in the proportion of 1 to 4.

One hundred and sixty-eight (168) blanks record absence of left-
handedness in the direct ancestry; including 51 (2G % + 2.j(/ ) individuals
to and including all the great-grandparents, 92 (40$ +52,/) to and
including all the grandparents, and 30 blanks (43 lefthanded individuals)
with statements, " unable to get information," or " so far as is known."
This set includes a fraternity of three lefthanded, including a jjair of
twins (Fig. 57). Of another lefthanded individual the statement is
given that the lefthanded character is considered a " birth-mark," the
mother having been obliged by reason of injuiy to right arm to use
the left during pregnancy. Another history, in which no trace of left-
handedness is said to appear back to and including the gi'eat-
grandparents, includes two lefthanded males among 9 other males
and 3 females, including two pairs of ordinary twins (Fig. 58). Still
another records an individual " lefthanded from birth," an extraordinary
fact.

This latter group of data admits of three interpretations : (1) absence
of hereditary influence ; (2) spontaneous origin of lefthandedness, which
may be thereafter transmitted by heredity ; and (3) the " normals "
may all be heterozygotes. Moreover, mild degrees of lefthandedness
may have escaped notice or may have been ma.sked or lost in later life ;
or the information may in a certain number of instances be inaccurate.
That it does not indicate absence of hereditary influence appeal's from
such pedigi-ees as the one shown in Fig. 59 where the direct ancestors
for two generations on both sides are normal. This could be carried
backward in time to any previous generation without violence to
Mendelian principles. This chart is Fig. 10 of my paper, " Studies in
Human Heredity," 1912. These three histories. Figs. 57, 58 and 59, as
also Figs. 60 and 61, suggest forcibly that lefthandedness cannot be due
to the absence of a factor. As stated in an earlier paper the antithetic
condition due to factor absence is probably' ambidexterity. This will
be further discussed below.

The data in charts Figs. 60 to 62 were supplied by Professor
F. A. Hodge of Winthrop College, South Carolina. Concerning the
penultimate fraternity of six of chart 62 the statement is recorded that
these children were " made to wear gloves on the left hand when infants

H, E. Jordan 73

to keep them from becoming lefthanded. Charts 60 and 62 both
contain a fraternity fulfilling expectancy from a DR x RR cross.
The following table summarizes the data for this group :

Histories

of fraternities imth

rightha

DiffetZ ancestry.

Left

Eight

Left

Right

2

5

1

4

1

4

1

3

1

3

1

3

1

4

1

6

2

6

2

12

1

8

1

3

1

7

1

4

1

4

2

4

1

4

1

6

1

4

3

3

1

3

1

5

1

3

1

4

1

6

1

3

2

4

1

7

1

3

1

5 '

1

4

2

4

1

4

1

8

1

3

1

4

1

5

I

10

1

6

1

3

1

6

1

5

1

3

X

3

2

5

2

6

1

8

2

3

3

5

2

4

1

3

1

6

1

6

2

4

2

4

2

S

1

5

1

4

2

5

1

3

2

4

1

3

1

4

1

4

1

7

1

3

2

3

2

3

1

6

—

—

Totals

90

321

or

1

: 3-56

The most probable assumption is that the parents involved are
heterozygotes. The sum ratio, 1 to 3'56, is very close to expectancy.
Sixteen fraternities actually show the 1 to 3 ratio ; and sixteen others a
1 to 4 ratio. Still other ratios are significantly close. This body of
data seems all but conclusive in showing the general recessive nature

74 Hereditanj Lcftliandednean

of the lefthandedness "determiner," and the general agreement with
Mendelian hiws of inheritance.

Charts Figs. 63 to G7 present striking histories from the foregoing
group. In the first four the ancestors to, and including, the grand-
parents (Fig. 64 ; also gi-eat-gi'undparents) are said to have been normal.
The immediate parents may be presumed to be in the simplex condition
(heterozygous dominants). These four pedigrees show unmistakeably that
lefthandedness is due to a positive factor (presence of a "determiner").
The reported incidence of lefthandedness in the several fraternities
defies reconciliation with strict Mendelian principles'. Similarly,
pedigree Fig. 67. Here the entire childship of a double lefthanded
mating is reported ntiniuil. This liistory, as also many other facts, show
that righthandedness also depends upon a positive factor. Chai'ts 63
and 67 are directly contradictory, not only with i-eference to each other,
but also as concerns numerous other pedigrees and the princijjles of
lefthanded inheritance (Mendelian) deduced from them. These will be
iinther discussed among exceptions considered below.

Ciiart Fig. 68 is from the parent-child group. It is especially
interesting in fulfilling the Mendelian expectation of a DR x KR, the
probable cross.

Chai'ts Figs. 69 to 74 ai'e tlie most important and most extensive
among the "collateral" group noted above. Concerning the lefthanded
male parent of 71 it is rect)rded that he "bats leftlianded in base-ball."
The fraternities of Figs. 69 and 74 give the Mendelian ratio ioi' the
most probable crosses involved, namely DR x DR, and the remaindi'r
are not seriously at variance with exjDectanc}' in view of the pi-obable
ancestry.

Chart, Fig. 7"), was contributed by Professor F. A. Hodge, Winthrop
College, S.C. ; and Fig. 76 by Mr Frank J. Sconce, of Fairview, Illinois
(filed at the Eugenics Record Oftice, Cold Spring Harbor, Long Island).
Botli histories demonstrate the recessive nature of decided lefthanded
bias, and leave no doubt regarding the general valency of Mendelian
laws in hereditary lefthandedness. Chart Fig. 77 (Prof. Hodge) appears
to countervail this deduction ; but the reported lefthanded parents have
but a mild bias, as I have learned from subsequent inquiry, since " they
write with the right hand, and do only certain things with the left
hand." Complete recession, under the circumstances, would hardly be
expected. This pedigi-ee was charted as Fig. 32 of my " Studies in
Human Heredity."

' The possibility is suggested that lefthandedness (and ambidexterity) may be degrees
of the same condition due to an "inhibitor" to righthandedness.

H. E. Jordan 75

Chart Fig. 78 was given to me by Professor J. P. Campbell, of the
University of Georgia. It shows peculiarly well the inadequacy of only
the dii-ect ancestry in a study of hereditary lefthandedness and the
hereditary influence and significance of collateral ancestry, and suggests
tlie rarity of " pure " strains with respect of dexterity. Also, it is of
especial interest in giving a pair of duplicate twins, only one of which is
lefthanded (and both " cross-eyed "). This condition is the exception of
what usually obtains in the case of such twins. It suggests more forcibly
perhaps than any othei' fact the verity of " degi'ees of bias " to use of
hand.

Chart Fig. 79 is supplied by Mr J. H. Green, of Clitlon Forge,
Virginia. It is a fau-ly complete five-generation history of a large
family in which appear both lefthandedness and a tendency to twinning.
Nine pairs of twins appear; one member of pair 5 is lefthanded; but
these are not identical twins. The other members of this fraternity are
another pair of twins ; and the lefthanded condition appears in the
expected ratio for the DR x DR cross. Coincidence of lefthandedness
and twinning in the same fraternity is frequent ; but no very likely
explanation suggests itself regarding causative relationship. The
explanation of lefthanded duplicate twins however lies most probably in
the ancestral presence of both conditions ; hereditary twinning would
then produce a lefthanded pair when the determiner for lefthandedness
was coincidently present in the case of a dujjlicate set (c£ Figs. 22 and
57). The complete absence of lefthandedness in the product of mating
^ X X J' Y is interesting as suggesting a " pure " extraction from a
" tainted " stock.

A summary of the 79 histories charted gives a proportion of 173
lefthanded males to 14.3 females. This result is the opposite of the one
usually recorded, namely, that females show a heavier incidence of left-
handedness. My earlier studies gave an approximate equality ; and the
discrepancy here noted is not sufficiently great to have significance as
contradicting the general conclusion that males and females are equally
" susceptible " to lefthandedness.

Discussion.

No attempt has been made in the foregoing to explain contradictions
and exceptions. In view of the present study, and of two earlier ones,
no one, I believe, will seriously dispute the conclusion that lefthandedness
is hereditary. That it follows in inheritance Mendelian principles, a

76 Hereditarji Leftlumiledness

number of apparently serious contradictions may peiliaps still give some
gi-ound for question. However, the main mass of data points to strict
Mendelian hereditary conduct. The proportion of 1 lefthanded to 1^
righthanded obtained in both of my earlier studies when all histories
were included cogently suggests the operation of Mendelian laws, when
account is taken of the fact that the majoi-ity of the crosses were either
DR X Dlt (1 : 3) or DR x RR (1:1). Moreover, a considerable number
of histories gave approximately the expected proportions for such
crosses, where the most pi'obable condition of the [)aients was indicated
by the ancestry.

Major C. C. Hurst, on the basis of a study of a number of pedigrees,
confirms my conclusion that lefthandedness is a Mi'ndelian recessive'.
Dr E. Stier's pedigi'ee.s of double lefthanded matings (6 cases) force the
same conclusion, when allowance is made for occasional imperfection of
dominance or slight degi-ees of bias. In no instance are all of the
offspring lefthanded ; but with one exception the majority are left-
handed.

My present study gives additional, and more complete, confirmation
of my earlier conclusion. A general survey of these charts permits
no doubt that lefthandedness is hereditary. This conclusion is further
suggested by cases of duplicate twins, where both members of the pair
are lefthanded (Figs. 22 and 57 — exception Fig. 78). When therefore
lefthandedness appears in a fraternity, at least one of the parents is
simplex for the lefthandedness factor, frequently perhaps both. Sum-
marizing the 69 pedigi'ees in the group where the ancestry is said to be
normal, and where the fraternities contain four or more individuals,
the ratio of lefthanded to righthanded is as 1 : 3\5G (page 73). This
is so close to Mendelian expectancy for DR x DR crosses as to furnish
almost a demonstration of the actual fact. The slight preponderance of
righthandedness is undoubtedly to be accountetl for by the occasional
DD X DR crosses with possible imperfections of dduiinance. Moreover,
the inclusion of sixteen 1 : 3 ratios, and the same number of 1 : 4 ratios
still more firmly supports the conclusion. Likewise, the ratios of the
other two tables above discussed and the average of all, 1 : 3'31.
A ruimber of the pedigi'ees here charted give also the exact ratio
expected on Mendelian assumptions.

Again, double lefthanded matings give complete lefthanded frater-
nities (Figs. 5, 16 — almost, 75, and 76), as expected from pure recessive
parents. The apparent contradiction. Fig. 77, is to be explained on

' Likewise more recently Professor Francis Ramaley (Am. Nat., Dec. 1913).

H. E. Jordan 77

the basis of faulty record, since later inquiry elicited the information
that the " lefthanded " parents were only slightly so, and that they
wrote with the right hand. The very fact of the ability to write well
with the right hand indicates slight bias, which would be expected
to be only imperfectly conformable with strict Mendelian formulae-
Chart 67 may perhaps legitimately be similarly interpreted, in the
absence of more definite information. The contradictions, Figs. 5, 39,
63, 64, 65 and 66, still remain. Here presumably " normal " parents
have all lefthanded children.

Already in my first study I was led to the idea of " degrees of bias."
This phenomenon has only become more clear in my second and the
present studies. How shall we account for the origin of degrees, and
what is their significance in inheritance ? This discussion further
involves a consideration of the apparent dominance of lefthandedness in
certain strains (Figs. 5, 8, 12, 14, 22, 46).

Both phylogenetic and ontogenetic facts show that the ancestral
condition with respect of use of hand was ambidexterity. Anthropo-
hjgical data point in the same direction. Both lefthandedness and
righthandedness represent variations from the ambidextral condition.
The antithetic condition to both (i.e. the one characterized by nuUiplicity
or absence of determiner) is natural ambidexterity.

The spontaneous origin of lefthandedness and righthandedness in
ontogeny suggests, gi'anting full validity to the biogenetic law, that
racially these conditions also arose spontaneously, i.e. discontinuously ;
not as the result of a slow accumulation of slight variations or fluctua-
tions— a mutation, not an acquired character now become hereditary.
The fundamental anatomic variation presumably inheres in a foetal
asymmetry of the cerebral blood supply producing probably an unequal
development (microscopic) of the hemispheres. This phase of the ques-
tion is discussed in my former papers.

The usual variation both phylogenetic and ontogenetic is toward a
righthandcd bias. Ancestrally those individuals that varied toward
a lefthanded bias came, perhaps, to some extent under the influence of
natural selection. Present lefthanded individuals would thus trace their
ancestry back to those earliest ancestors who escaped elimination.

The dominance of righthandedness over lefthandedness is also in
accord with the principle of progressive evolution through discontinuity.
There can be little doubt that under present conditions of community
life, dexterity gives considerable advantage over sinisterity or even
perhaps ambidexterity. Indeed righthandedness may easily be conceived

78 HercdUanj Lr/f handedness

of as having contribntcd t-<i a cinisidcrable degree to the attainment of
the present level of civilization. The telcological significance of the
dominance of righthandcdness is obvious. However, the cause of
prepotency is here as ob.scui'e as elsewhere. The evidence on this point
is conflicting; now ancestral conditions (racially older) seem to be
dominant, m other instances the new, and again the more intense, are
prepotent. Progress by discontinuous variation would seem to be
possible only through the prepotency of determiners signifying improved
adjustment.

Genetic studies of bias toward use of one or the other hand promise
to throw .some light on the (piestion of the stability of "determiners" or
" genes." Degrees of prepotency of characters, so strikingly evident in
the case of the heredity of use of hand, suggests considerable lability of
genes.

Elucidation of the whole matter proceeds with inneh plausibility
from the recent suggestion by Miss Elderton, that dominance may not
attach to a character but to the individual with the character, "i.e. that
the same character can in certain individuals be dominant and in others
be recessive," p. 34. Whether by reason of a numerically })re])onderant
variation towards dexterity relative to sinisterity, or a numerically equal
but more decided bias in that direction, righthandcdness early came to
be the numerically predominant condition, now present approximately
in the ratio of 4 to 1. Under later civilization lefthandedness became
an increasingly greater handicap. Thus individuals in whom this
handicapping character of great intensity was dominaul may have been
eliminated through differential selection. In this wise only recessive
lefthandedness of high degree has come to remain almost exclusively,
and the ideas of dominance and recession have come to be attached to
the character itself. Less extreme degree of lefthandedness due to a
greater power of adaptability and training may thus have to some extent
remained of a dominant nature.

Thus the parents in charts 63 to 66 may possibly have been left-
handed individuals of so slight degree as to have escaped notice as such
in childhood or forgotten at maturity, but of dominant nature ; then,
whether duplex or simplex, all the children would be expected to be
lefthanded. Likewise in pedigrees like Figs. 67 and 77 the parents
may be simplex dominants (for lefthandedness), thus of mild degree,
and their naturally lefthanded offspring of slight bias may by slight
or even unpremeditated training have been made to acquire right-
handedness.

H. E. JOKDAN 79

In cuiiclusion; the data with reference to hereditary lefthandedness
are overwhelmingly in accord with Mendelian theory. Certain real
exceptions appear; those, however, are no more forbidding than those
relating to certain other human characteristics, and readily permit of
complete reconciliation by aid of the suggested hypotheses of degrees
of intensity of bias, and mild dominant and intense recessive strains
of lefthandedness. Whether this interpretation of " exceptions " be
thought admissible or not, the mass of strictly conformable evidence
and data is still so considerable as to compel the conclusion of the
general validity of Mendelian law in the inheritance of lefthandedness.

A Note on Twinning.

Davenpoi't has called attention to the inheritance of this tendency,
and illustrates the phenomenon by two suggestive pedigrees recorded
by Stocks and by Wakley. Dr James Oliver has more recently
published abbreviated pedigi'ee charts of 28 sets of twins, showing an
hereditary tendency to twinning. The data are perhaps not yet of
sufficient quantity to warrant .deductions as to laws of twin heredity-
Charts 79 and 80 show perhaps simply that t^vins in one's ancestry
gives a fair prospect of twins in one's progeny. The fact that " normal "
parents may have twins suggests that the factor (or factors) for
twinning is a Mendelian recessive. This is further suggested by the
circumstance that a normal (A) x twin (B) cross produced a fraternity of
10, all normal (Fig. 79). However, the pedigrees cited by Davenport
seem to leave little doubt that the twinning tendency is in some
instances " dominant " ; probably a complex of factors is involved.
Moreover, contrary to Dr Oliver's data, which shows a preponderance of
female to male twins to the extent of 2 to 1, chart 79 shows a prepon-
derance of males in almost the reverse ratio, 7 females to 11 males.
The same deduction is indicated in a pedigree (Fig. 80)' supplied by
Mr R. G. Reaves, of Greenville, Tennessee, in which there arc 14 pairs
(1 with sex unknown) including 18 males and 8 females (6 male pairs,
1 female pair, and 6 bi-sexual pairs).

1 This is of conrse only a partial pedigree in the earlier generations ; it includes,
however, all known pairs of twins ; the total number of descendants of the first mating
charted is approximately 1100. Childship (M) fulfils Mendelian expectancy for the
probable cross, DR x ER.

80 Hereditarji Lc/f/iandedness

Twill birth is presumably simply a lesser degree (if multiple-birth,
characteristic of lower mammals. In general, the higher the degree of
development (specialization) the fewer the individuals at a birth. In
horses twins are rare ; in certain sheep they are the rule. Fewer
individuals at a single birth implies evolutionary advance. From this
viewpoints tiie suggested recession of human twin birth to the more
normal birth becomes intelligible in terms of the dominance of a derived
or advanced, over a racially older, condition.

The pedigrees given by both Oliver and Davenport show trans-
mission through males only. In both of my own histories, the trans-
mission is more frequently through the female line.

On the basis of the experimental evidence from invertebrates,
Amphioxus, and amphibia identical (monochorial ; duplicate) twins are
commonly explained in terms of a relatively independent development of
accidentally separated blastomeres, at the two-cell stage of development.
The stimulus or causative agent to separation (or " budding " of em-
bryonic disc as in armadillo — Patterson; or "fission," — Assheton) may
conceivably be either mechanical or chemical (nutritive — Patterson). If
the former, an inheritance of the tendency would hardly be expected.
The latter circumstance seems the more probable. The chemical factor
for disjunction might inhere either in the male or the female gamete.
On this basis heredity of duplicate twins becomes intelligible.

But occasionally bi-sexual (heterosexual or ordinary) pairs of twins
occur in such pedigrees {vide Oliver, Davenport, and Figs. 79 and 80).
Ordinary twins are commonly supposed to be due to the practically
simultaneous ovulation of both ovaries, or a double ovulation on the
part of the functioning ovary. Such tendency may well be hereditary,
but it could obviously show itself only in females. It is inconceivable
how the spermatozoon could exert influence on the ovary such as to
stimulate to double ovulation. Inheritance on the above explanation
would have to follow an alternation of generations where males inter-
vened ; i.e. only females of such a pedigree could determine twins of the
ordinary type. In view of the several pedigrees here involved, it nnist
remain doubtful whether such is the case, though pedigrees 79 and 80'
are suggestive of this condition. Both ordinary and identical twins
appear in the same pedigree, and both conditions seem hereditary
according to similar principles, the tendency for both being apparently
transmitted either by male or female. It seems in the light of known

' Twin pairs of the same sex are known to be "identical" only in the instances
so specified; regarding the remainder the information was uncertain.

H. E. Jordan 81

pedigrees of human twiimiiig that the exjjlaiiation of ordinary and
duplicate twins is probably not as simple a matter as has been supposed.
It would appear that the sperm is as potent as the egg, in determining
twins, either ordinary or identical.

LITERATURE CITED.

AssHETON, Richard. " Fission of Embryonal Area in JIammals ; Summary."

Journ. Roy. Micr. Soc. Oct. 1913.
Davenport, C. B. Heredity in Relation to Eugenics. H. Holt and Co., N.Y., 1911.
Elderton, Ethel M. On the Marriage of First Cousins. Eugenics Laboratory

Lecture, Series V, Dulau and Co., London, 1911.
Hurst, C. C. " Mendelian Heredity in Man." Eugenics Reoiew, April, 1012.
Jordan, H. E. "The Liheritance of Lefthaudedness." American Breeders Maga-
zine, Vol. n. Nos. 1 and 2, 1911.
. "Studies in Human Heredity." Bulletin of the Philosophiad Society,

Univ. of Virginia, Scientific Series, Vol. i. No. 12, July, 1912.
Oliver, Jame.s. "The Hereditary Tendency to Twinning." Eugenics Revieu', April

and July, 1912.
Patterson, J. T. "A Preliminary Report on the Demonstration of Polyembryonic

Development in the Armadillo." Anat. Anz. Bd. xli. No. 13, 1912. (Full

report, Journ. Morph. Vol. xxiv. No. 4, 1913.)
Stier, Ewald. Untersuchungcn iiber Linkshdndigkeit. Gustav Fischer, Juna, 1911.

Journ. of Gen. iv

JOURNAL OF GENETICS, VOL. IV. NO. 1

? t ? i

i i i . 1 . t

! I I I I . i . ,

? rf ? ? r— I— n t ^ rf '"1— <?

I I I I I I I I I I

III! I I I I I I !

i (4)* i (1) ? (3) t (}) f i m i i i i ^

(1) (2) (3) (4) (5) (6) (7) (8)

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I I I I I i ! I I I I 1 I I !

(13) (14) (15) (16) (171

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. III! I

twm.s twins ^ (3) 9 9 1 9 i (.^') S 9 6 i 9 9

(22) (23) (24) (25) (26)

1^ I III I III I i I

6 f 9 9 6 t (8) c? rf <? i m rf (4) 9

(35) (36) (37) (38) (39)

1-3 Lpfthaiidediiess trace<able five generations in the direct line. (Three histories.)

8—16 Lefthaudedness appearing in parent and child. (Korty-eight histories.)

23—36 Lefthandedness appearing in great-grandparent and child. (Fourteen histories.)

41—56 Lefthandedness appearing in grandparent, parent and child. (Sixteen histories.)

* The numeral gives the order in the fraternity ; the total number of indi>i

FIGURES 1—46

?-

r'

I I
(9)

?

I

6
I
f

I

i (3)

(18)

1 1

I I
? ?

i

I

I I

n

r-'^i n

I I I
(? c? ? ?

(10)

I
?

I I

t? (? ? i 9
(19)

I i I

S 6 S

(20)

— 6

I I

i i

j_

~i — I
I I i I

6 6 S f
(11)

t

I
?

I 1 1 1 r-

I I I I I I
rf ? ^ ? (? c?

(21)

_1.

I I

? ?

(12)

i i I

I
?

(28)

i

I

unknown

i ?

I I

I I
I

^ (4)
(29)

I !

S 6

6

I

? (2)

6

I

(30) (31)

I

I
i (1)

(41) (42)

J Ambidextrous

I

^ (5)

I 1

I I

i i

I

i

I
i (2)

(43)

I

s

I

(32)

I

9 rf ? c?

(33)

""I
I
?

I I

I I I

(? ?

9
(44)

i

I

* (3)
(45)

9-— r-c?

?

I
c?-T-9

n

(34)

(46)

4 — 7 Lefthandedness appearing in four consecutive generations. (Pour histories.)
7—22 Lefthandedness appearing in great-grandparent, parent and child. (Six histories.)
7— 40 Lefthandedness appearing in great -grandparent, grandparent, and child. (Four histories.)

s is not recorded. Normal consorts are generally omitted in the charts.

JOURNAL OF GENETICS, VOL. IV. NO. 1

I
i

f (1)*

(47)

I

i (1)

(48)

I 1 1

I I I

i i t

I

i

i (1)

(49)

?

I

I
I

I
6

i

I

-it

III!
^ i i i

(50)

f

I

(51)

(52)

I I

9 — T — i
I
?

I I

twins

I I I I
twins

I
i

I
— ^ 6

(58)

: I I I

^ rf (? c?

I

^ 9 —

I

I I

(59)

I —

I

6

I

T"
9

1 I I
? 9 9

r

1

1

1

i

6

9

1

6

1
1
6

1
1

6

(61)

I I
? 9

I —

I

9

I
6

i i f f-

I I I I I

-9 9 9 9 cJ

I I
6 S

(62)

41-56 liof'thaiidojiiess aiipc^iring
* Tlio inunoial give.s the ureter in the fraternity; the total number

in grani
of indi

FIGURES 47—69

?

I

I

t (4)

(53)

I

i

I

-6

I
(54)

I
i (1)

(55)

-<? 9-

?

I
?

I
T (1)

(56)

I

-6

4-

I I I

twins

(57)

-<? 9-

I

I

he?

I I I

f 9 (?

I

(?-

1 ! I
c? <? 9

I I

9 9-T-<?

I I
-? 9

I I I
16 6

(60)

I I
9 9

-1 1 1 1

I I I I
6 6 6 6

-^-6

I

! I

f i

(63)

-6 9-

I

r-+-n

I I I
9 9 <?

(67)

I

I

f i

(64)

-6 9-

I
I I

I
-6

i i i

(68)

I I

I

^6

I

i

I I I

(65)

I —

I

?

I

r-+-

I I

i ?

(66)

I — n

I I

I

I I I

i 9 9

(69)

mt, parent and child. (Sixteen histories.)

.Is is not recorded. Normal consorts are generally omitted in the charts.

JOURNAL OF GENETICS, VOL. IV. NO. 1

n

i 9-

I I
6 ?

(70)

-6 9-

I

I I I I

9 i 9 9
(71)

1

i 9-

I

9 f 6
(72)

I I
? 9

\ T~7

6 6 9

9~-r-S

! I I

i 3 i

-] —

i

1

i

1

1

1

9

i
f

-r
1
?

1

i

__

-r

1
1

I

i i I I

i f i i

(76;

I I

I I

o o t

(77)

I ! I

o o o

ix!.

I I I I I I

c? ? ? ? 9 9

I
6

* Cf. text, pp. 74 .-

FIGURES 70—78

?-- r-(? ?

9-^-^*

— 6

1 1 1 1

A 1 1 1
9 f 6 S

1

i

—^^

1

1

1
6

* Ambidextrou^

(74)

9-T-(? ■?■

-cJ 9-

I
I \ I 1 1 \ 1 1 1

I I I I ! I I I I

(75)

I

■t-

I

6-

I I I I
-9 S 6 6

I

6

died young

6 6-

identical twins
(cross-eyed)
? 9 9 (infant of a few weeks)

I I

9—r~6

I

I I I

f i 9

I

I I
9 9

I i
6 i

I

(78)

JOURNAL OF GENETICS, VOL. IV. NO. 1

■6 9 9 6 >i

n^

4^99319

->J

-i 9 i^_ 6 6 6 ^< 9 d .i 9

9 J

???c^9? 9 i 9 6 6

I 1119 t

9 i l_rf I I

4) (,'i)(y) fi r

9 9 <J

(4)

9 6 f

I

9 (?

rr

I I

6 9n

I

I

~i ( 1 ^ I 1 r

6 6 9

9 9 ^i

I llllllllcJJ?

9 9 96166fi9 -

9 9

f 6 9 6 6

I

9

* Am

FIGURE 79

-6 d 9 9 6 6 9 6 6

? 9

6 6 6 6 6 6

I
(8) 9

9 6

I 1 — — I — ~[

I I I I
9 9 6 6

ictroiis.

JOURNAL OF GENETICS, VOL. IV. NO. 1

'i—^d

6 6 S i S

01)
I

"f.ivoured each otlier,"
one (lied at, .■$ nicintlis,
illicr died .it, |;{ niontlis

FIGURE 80

i S 9 V-^t 9(^?9,^9J9(J

9 (J J,

n

9 cJ died at birth

A PECULIAR NEGATIVE COilRELATION IN
OENOTHERA HYBRIDS.

By GEORGE HARRISON SHULL.

Station for Expenmental EuoUition, of the Carnegie Institution
of Washington, Cold Spring Harbor, Long Island.

With all the careful attention which has been given to the
Oenotheras by students of genetic phenomena, there is no other group
of organisms in which these phenomena seem so far from a satisfactory
interpretation. Fundamental difficulties are encountered whenever
attempts are made to apply to the Oenotheras rules of genetic behavior
which are readily demonstrated in other groups of 02'ganisms. Equal
confusion has arisen by the application of genetic experiences with the
Oenotheras to species in which typical Mendelian phenomena appear.
A hereditary mechanism must exist in Oenothera fundamentally
different from that which distributes the Mendelian unit-characters.
It becomes increasingly clear that the data on record are inadequate for
the discovery of the essential features of this mechanism. It is con-
ceivable that different mechanisms for the distribution of the hereditary
characters may exist in different sections of the genus and that this
may partly account for the slowness with which a comprehensive view
of the genetic phenomena in this field has been made possible. Under
these circumstances there is demanded a large amount of purely
inductive work by the strictest pedigi-ee methods and individual
analysis. Before we can hope to explain the phenomena we must
know what phenomena are presented for explanation. As a modest
contribution to our knowledge of the genetic facts in Oenothera which
need interpretation the following data are presented.

Early in June 1912 I received from ])r. A. F. Blakeslee, Storrs,
Connecticut, three rosettes grown from unguarded seeds of Oenothera
rubricalg.r Gates, the seeds having been received by him from
Dr. R. R. Gates, the discoverer of this particularly beautiful form.

6—2

84 Negative Correlation in Oenothera Hybrids

The rosettes hail the general in.irph<ilnj,neal teatun^s (it my O. nibri-
iiervis foriiis, but the leaves were darker gi-een and of more shining
surface. The buds were more slender and more nearly terete, but the
capsules were long and decorated, when young, with the 2-4 deep-red
longitudinal bands usually seen on immature rubrwervis-cnTpsuh-^. The
buds were brilliantly red-pigmented throughout, except on the ovary
and the distal portion of the free tips, which were gi-een. The stems
were also brilliantly red-pigmented, as described by Gates. All three of
the plants were of the same type, and it seems probable that they were
true 0. rubricalyx G-Ates, despite the fact that they aime from unguarded
seeds under circumstances doubtless favorable to crossing with many
other Oenotheras'. As my results in breeding these plants differ
strikingly from those ivported l)y Gates, the possibility of the hybi-id
nature of my original plants of this form must be kept in mind ; but it
must also be kept in mind that even if these three plants were hybrids,
they must each have been produced by the union of only two germ-cells,
and as I have used in the self-fertilization and in all the crosses described
below only one of the three original plants, any complication fmm their
possibly hybrid nature is reduced to the lowest possible minimum. Such
a possible hybrid origin will not detract in the least from the interesting
con-elations with which this paper deals. It should be added that in
making reciprocal crosses generally, I have used in both crosses the same
pair of individuals, so that no unsuspected complications can have been
introduced by genotyj)ic differences in indi\iduals wronglv assumed to
be identical because they were phenotypically alike. This is a detail
of technique of the utmost importance ; it will be found that the
genetic problems in Oenothera are sufficiently complex when the
elements which enter into the related crosses are reduced to the lowest
possible terms.

Oenothera ?v(6/'tca/^.*; appeared in 1907 in a mixe<l progeny consisting
of 112 otFsj)ring of four self-fertilized ruhrtnervis-'pl-Ants grown at Woods
Hole the jjreceding year, from unguardeil^ .seeds received from de Vries

' Several features of my plants suggested a relationship to 0. yrandijiora, particularly
the rather lax rosettes, strong red spotting of the young rosette-leaves, and the develop-
ment of buds more slender and rounder than in my 0. rubrincrvis cultures, but as none of
the characters presented by the offspring showed accentuated resemblance to 0. grandijlora,
I doubt the reality of this suggested relationship.

- Gates does not state that the seeds received from de Tries were from open pollinations,
but the experience of Ooio^/it-rrt-students with niiriJifn'is-culturcs, makes probable no
other hiterpretation of the occurrence of 2 0 iMniarcktuiia and 1 0. oblnnga among the
45 plants which included the n/fcriHi'm's-parent of the original 0. rubricalyx.

G. H. Shull 85

(Gates, 1918 b). The original riib7-icali/x-Y)\ant, self-fertilized, yielded
9 0. ruhricalyx, 1 ruhrinervis, 2 undetermined'. Three self-fertilizations
of these r uhricaly x-ST^Qcimens produced in the next generation 57
ruhricaly.r and 22 ruhrinervis, showing in one family of 44 plants a
ratio 3 : 1, and another family grown from seeds of an open-pollinated
rubricalyx-Tplant in the same generation, yielded 71 rubricaly.r, 38
ruhrinervis and 4 doubtful-'. (J wing to various misfortunes the progenies
from several other self-fertilizations could not be classified, but both
ruhricalyx- and ruhrinervis-T^\-Anis were apparently present. From these
results Gates concluded that ruhricalyx originated as a heterozygote
which differed from the parent in a single, dominant, purely quanti-
tative, Mendelian character, and strangely enough at the same time
concluded that it would always split into ruhricalyx and ruhrinervis
(Gates, 1911).

The small number of plants which reached maturity in Gates's
pedigrees aroused the desire to test more thoroughly the apparently
uniijue behavior of the r«6ric«%i'-character. Since my experiments
with this form were undertaken, Gates (1912) has announced the
discovery of a " homozygous " individual of 0. ruhricalyx, which breeds
true to this character, thus proving the incorrectness of his conclusion
regarding the continued splitting of the ruhricalyx-T^vogemes, but on
the other hand strengthening his assumption that the ruhricalyx-
character is Mendelian in inheritance. His oft repeated emphasis of
the view that 0. ruhricalyx differs from 0. ruhrinervis in a purely
quantitative character which acts as a monohybrid Mendelian dominant
over the ruhrinervis-type of pigmentation will have a special interest in
relation to the results described below. In a paper published since
this was written Gates (1914) wavers between the treatment of the
ruhricalyx type of pigmentation as a Mendelian and as a non-Mendelian
character. His most positive declarations on the subject are that it is
non-Mendelian ; but if he sincerely holds to this conviction it is strange
that he should continue to treat the genetic behavior of this character
as if it threw valuable sidelights on Mendelian phenomena.

During the past season (1913) I have had five pedigrees derived
from one of the three individuals of 0. ruhricalyx grown by me in 1912.
These were 0. ruhricalyx self-fertilized, 0. ruhricalyx x ruhrinervis,

' These two undetermined plants are listed in one place as 0. ruhricalyx. See Gates,
1911, Table II, p. 365.

- In the same place (Gates 1911, Table II) this family is erroneously indicated as the
product of a self-fertilization and the four doubtful plants are included under 0. ruhrinenns.

86 Negative Correlation in Oenothera Hybrids

0. ruhrinervis x ruhricalyx, 0. ruhricalyx x Lamarckiana and 0. La-
marckiana x ruhricaljx. All of these were sown on the same day,
grown under the same conditions, potted, described and photogi-aphed
at approximately the same ages, and set into the field in a<ljacent rows
on the same day. Each of these families will be considered under a
separate heading.

Oenothera ruhricalyx Gates.
Pedigree No. 11410(1) x Self = 1231.

Seeds sown January .30th, 1913, germinated in 16 — "20 days and
yielded 119 plants, which at 10 weeks of age were clearly divisible into
two groups in respect to moi'phological characters, and into two inde-
pendent groups in regard to the pigmentation of the leaves. These
four groups may be briefly characterized as (ii) the ruhrinervisASke
group, (b) the nanella-Wke group, (c) the spotted group, and {d) the
unspotted group. Two plants died unclassified. The remaining 117
were distributed as follows : (ac) 59, {ad) 48, (he) 8, {hd) 2. These
categories may be briefiy characterized as follows :

Plants of the (a) group containing 107 individuals, or 91"45 per cent,
of the entire family, had rather lax, ascending rosettes of relatively
narrow leaves, resembling those of 0. ruhrinervis, but in general a little
darker green and with more reddening of petioles and midribs in the
older leaves, the spotted sub-group (c) usually being a little more
strongly crinkled and a little more prominently dentate toward the
base of the blades than the unspotted sub-group (d), as seen in the
upper series of plants in Plate V. These may be compared with pure-
bred 0. rubrinervis-vosettes of similar age, which are shown in the lower
series on the same Plate. The adult plants all had the tall, wide-branching
form usually seen in vigorous strains of 0. rnbrinevvis, with the
characteristic long capsules of that species, but the buds were as in
the parent, relatively a little more slender than in my long-controlled
sti-ains of 0. ruhrinerms.

In the following table, bud-measurements of these ruhricalyx-Yi\fmiii
both red-stemmed (r/c), and pale-stemmed (ad), may be compared with
corresponding measurements from four pure 0. ruhri/iervis-pedigrees
taken at about the same time (Aug. 1) and under similar conditions.
Each measurement entered in the table is an average from fi\e buds
taken at random from five different jilaiits in the indicated pedigree. All

G. H. SnuLL 87

nieasuiements are in centimeters and were made with a micrometer-
caliper by my scientific assistant, W. F. Friedman, to whose faithful
work and painstaking care it is a pleasure to give this grateful
recognition.

0.

riihrinervH

0. rul

riealyx

Character

Ped. No.

/ —
1213

12U

1215

1216

Av.

1231 ((/(■)

1231 ((If/)

Length of ovary ...

114

113

107

1-17

1-13

i-oi;

l-0(»

Thickness of ovary •

0-33

0-32

0-32

0-31

0-32

0-30

0-28

Length of hypanthium ...

3-03

3-93

3-55

3 03

3-53

413

3-77

Thickness of hypanthium-

0-31

0-28

0-25

0-28

0-26

0-24

0-24

Length of coue

4-21

4-43

3-95

4-00

4-16

4 -CI

4 -07

Thickness of cone^

0-81

0-84

0-75

0-74

0-78

0-74

0-72

Length of free tip

0-59

0-63

0-6G

0-66

0-63

0-74

0 00

Length of anther

1-54

1-58

—

1-66

1-59

1-49

1-45

Plants of the (6) gi-oup were distinguishable by their closer rosettes,
caused by the relatively short, ascending petioles and nearly horizontal
blades. They were recognized at once as nanella-like rosettes, though
their longer, narrower, less crinkled leaves distinguished them strikingly
from the nanella-iorm derived from 0. Lamarckiana. The adult plants
proved to be of little more than nanella-stature (25 — 40 cm.) as shown
in fig. 1, but of very unique aspect, due to the broad-lanceolate,
acuminate, dark gi-een, nearly uncrinkled leaves. The buds were l<jng,
slender and the cones nearly terete. Gates (1914) reports the occuiTcnce
of dwarfs in his cultures of both 0. rubricalyx and 0. graiHliflora, and
their recurrence in some of the F^ families from crosses between these
two species.

The gi'oup (c) differed from the (fZ) gi-oup in having conspicuous red
spots on the dorsal surface of the leaf-blades, as shown in the accom-
panying Plates. As Gates had found self-fertilized rubricalyx yielding
progenies containing both rubricalyx and rubrinervis, I at once infei'red
that I was getting the same result, and that the spotted rosettes belonged
to the rubricalyx- and the unspotted rosettes to the rubri^iervis-type.
This did not prove to be true, however, as the entire (a) group, both
spotted and unspotted, had the intensely pigmented hypanthia and bud-
cones characteristic of 0. rubricalyx. In the adult stage the spotted and
unspotted gi-oups, (c) and (d), were definitely differentiated from one
another in only one feature, namely, in the pigmentation of the stems.
The group grown from spotted rosettes, {ac), had intensely red-pigmented
stems, the pigment being particularly conspicuous about the base of the

' Greatest diameter.

- Least diameter.

'■' Greatest diameter parallel with sides.

88

Negative Correlation in Oenothera Hi/hrids

central spike and on the upper lateral branches. They were in this
respect like their parent. .The nnspotted group, on the other hand, had
the stem.s only slightly reddened, with the upjier part of the main-stem
and upper laterals not conspicuously pigmented with anthocyan. In
the dwarf group, (b), only six plants bloonu'd, all belonging to the
sub-group with spotted rosettes. Five of them had no anthoeyan in
the buds, and one had the pigmentation of (J. rubriculi/.v. All had
strongly reddened stems.

Fig. 1. A dwaif plant witli dark red steins and green buds from self-
fertilized (>, rubric(th/.v. (The included scale of inches was drawn
from a rule photographed on the same negative ; 1 inch = 2'5 cm.)

Gates has not mentioned red-spotting of the young rosette-leaves as
a characteristic of 0. rahricalyx, but on the other hand he now includes
this character among the features which dift'eri'Utiate 0. (/randiflura
from 0. rubricalyx (Gates, 1914). The fact that half of my rubncalyx-
plants had strongly spotted leaves may perhaps mean, therefore, that
my original »'«6nc'«i^.r-plants w'ere hybrids. Another possibility may
be suggested. Gates (1914) characterizes the rosette of 0. grandijlora

G. H. Shull 89

as having "pale red blotches " ; in my cultures that species had deep red
blotches. There can be no doubt that anthbcyan production is strongly
influenced by sunlight and temperature, and possibly other environ-
mental factors, which differ in different localities. The deeper red spots
on 0. yrandiflora in my cultures might be due to genotypic differences
between Gates's material of that sjjecies and mine, but it seems rather
more likely that they indicate a condition in my cultures favorable to
the intensification of the anthocyan colors. It is conceivable that a
form which does not possess red spots at all under one condition may be
strongly spotted under some other condition. This is directly illustrated
by the spotted plants themselves, for the spotting is an evanescent
character, which C(_impletely disappears as the rosettes grow older. The
occurrence of spotting in the young rosette-leaves in my cultures of
0. ruhricalya-, does not necessarily prove, therefoi-e, that my plants are
hybrid derivatives of 0. ruhricalyx instead of that species itself,
although on certain giounds the assumption that they are hybrids may
seem to be the more tenable hypothesis.

As the ruhricalyx-^\a,\\ts in this pedigree were of two types with
respect to the pigmentation of the stems, it was thought possible that
such splitting might also occur in the homozygous strain discovered by
Gates, but a letter from A. W. Sutton, Esq., Reading, England, informs
me that all of the 0. rubnc(dyx-ph\i\ts from Gates's pure-breeding stock
have the brilliant red stems'.

Oenothera ruhricalyx x ruhrinervis F^.
Pedigree Nos. 11410(1) x 1123(8)= 1232.

The male parent was from a pure cross-bred strain of Oenothera
ruhrinervis which had been full)' controlled in my cultures in seven
consecutive generations, each of these ancestral generations having
residted from a cross between two typical ruhrinervis-specimens, of as
widely separate relationship as ray several cultures provided. All the
preceding generations had been uniformly typical 0. ruhrinervis with
the exception of an extremely small percentage of aberrant individuals
which have appeared fi-om time to time as characteristic mutants(?).

Seeds from this cross yielded 205 individuals of which 8 died
unclassified. These were, like the progeny from the selfed 0. ruhriadyx

' Seeds of tliis form are now being offered for sale by Sutton and Sons under the name
" Oenothera Afterglow.'

90 Negative Correlation in Oenothera Jlijhrids

(No. 1231), divisible into a spotted and an unspotted group, in the ratio
103:94. No naHe//((-piants were seen, and all were similar in morpho-
logical featui-es to 0. rubrine7-vis, as shown in the second I'ow of rosette.s
from the top of Plate V, though in the 10-weeks-old rosettes about
two-thirds of the unspotted group had somewhat broader, darker green
leaves than the remaining one-third. Later, however, this distinction
could no longer be seen ; the broader- and narrower-leafed groups were
kept separate throughout their development, but no distinction was
visible between them in the mature jjlants. With the exception of
three plants which were slightly divergent in characters of foliage and
branching, all were of the same vegetative form, and indistinguishable
in this respect from the («) group of selfed 0. yahnculyx. In pigmenta-
tion, however, a striking situation was presented. The unspotted
rosettes developed into plants of the .same type as the corresponding
group in the selfed 0. ruhricalyx family, having greenish stems and
brilliantly pigmented buds of the ruhricalyx-ty^e. The spotted rosettes,
on the other hand, produced brilliantly red-pigmented stems, with buds
of the Laninrckiana-iy^e of pigmentation, the cones being merely pink
in longitudinal bands of gi-eater or lesser width, and the hypanthia
green. No plant of the latter group had buds as strongly pigmented
as in 0. ruhrinervis, the male parent, and the minus variations ranged
to cones nearly, though not quite, completely free from anthocyan.

Oenothera ruhrinervis x ruhricalyx F^.

Pedigree Nos. 1123(S) x 11410(1)= 1233.

Seeds from this cross produced 152 plants which were likewise
clearly separable into a spotted and an unspotted group, but with no
other apparent distinction. Two-months-old rosettes are shown in the
second row frotn the bottom of Plate V^. The two groups consisted of
62 spotted and 89 unspotted rosettes, 1 having died unclassified. Plants
of both groups had red on the under side of petioles and leaf-blades,
especially when the latter were going into decline. Several plants
which had one or two obscure red spots or fine red specks, but 2)ro\ ed
on their subsecjuent development to belong to the unspotted gi'oup, are
included with that group in the ratio given above. One plant which
was marked on May 2 as spotted, was found two weeks later without
spots, and was transfened to the unspotted group, where its identity'
was lost. Among tiie adult ])lants one indivi(hial from the unspotted

G. H. Shull 91

group had the bud- and stem-characters of the spotted plants, from
which I infer that the original determination of the character of this
plant was the correct one, and it is therefore included with the spotted
plants in the above reckoning. Only 25 of each group were set into
the field to grow to maturity. All the adult plants had the branching
habit and long capsules characteristic of 0. ruhricalyx and 0. rubrinervis.
Eight in the spotted gi'oup had slightly broader, darker gi-een, more
strongly crinkled leaves than their sibs, in these particulars more
closely approaching the characters of 0. Laiiiurckianu, but their stems
and buds did not differ in pigmentation from those of the other
plants in the spotted group. One plant in the spotted group had the
buds colored as in 0. rubrinervis, but was so heavy in all its parts as
to suggest the likelihood that it was a triploid form. Its capsules
were notably thicker and shorter than those of its sibs. I have
no special record as to the color of the stem of this plant, but
as it stood in the red-stemmed gi'oup, it would probably have been
noticed if different in this respect from the rest of the group. All the
rest of the spotted gi'oup had brilliant red stems and Lamarckiuna-Mke
buds with pink cones and green hypanthia. The unspotted gi'oup, with
the one exception already mentioned, had greenish stems and the
typical r ub)'icaly x-co\ora,tion of the buds, i.e., with hypanthia and cones
uniformly and intensely red-pigmented, except on the distal portion of
the free tips of the sepals, which were free from anthocyan.

Oenothera nihriculyx x Lamarelciana F^.

Pedigree No.s. 11410(1) x 118(10) = 1234.

Of this pedigree I secured 123 plants which were again divisible
into spotted and unspotted groups, but the spotting was not so
pronounced as in the ruhricalyx-rubrinervis-hyhrids, so that it was
not quite certain whether the unspotted group was a natural group
or simply the minus-end of a single fluotuating series. The gi'ouping
of the young rosettes gave 97 spotted and 26 unspotted plants. None
of these rosettes closely resembled 0. Luniarckiana, being more lax, the
leaves having longer petioles and more tapering bases ; and they were
also more ascending than in the rosettes of 0. Lamarckiana. They
may be compared with 0. Lamarckiana in Plate VI, in which 0. rubri-
calyx-ro's.ettes are at the top, 0. Lamarckiana at the bottom, and the
present series of hybrids in the second row from the top. The foliage

92 Negative Correlation In Oenothera Ilj/hrids

in the spotted group was perceptibly darker green and the leaves
slightly broader and more crinkled than in the unspotted group.
As the rosettes grew older, a small number were noted, which were
differentiated from the rest in being rather coarsely crinkled, with the
obvate to oblong-dbvate blades abruptly contracted, with a slight
undulation, to a ti'iangular- winged petiole. The lea\es <if' these had
more numerous and more conspicucius red spots than their spotted sibs.
That this gioup grades into the more common type of spotted ])lants, is
shown by the fact that I separated only 6 out of the 9 plants which
subse(juent development pro veil to belong to the same natural group.
Fifty plants were set in the field, including 30 spotted and 20 unspotted
plants, the members of each of these groups being taken at random,
with the exception of one of the crinkled-leafed, strongly spotted plants
just described, which was discovered before the reservations were made
for the field cultures. Aside from this one individual, therefore, the
distribution of the different types in the field should accurately repre-
sent, within the limits of the piobable error, the composition of the
entire progeny. The adult plants were all of one type with respect to
general habit of branching and the long capsules (2'5 — 27 cm.), being
in these regards like my selfed 0. ruhricalyx. Among them were three
classes of individuals sharply distinct from one another, namely,

{a) 9 [27]^ with very dark, dull-red stems, and buds wholly free
from red pigment ;

(b) 5 [17] with brilliant red stems, and Ldinairkiinm-Wki- buds,
i.e., with pink cones and green hypanthia; and

(c) 36 [79] with greenish stems and rubrical yx-hiuh, i.e., brilliant
red hypanthia and cones.

It would have been desirable to make an actual determination of
the relative amounts of anthocyan in the stems of groups (ff) and (b),
but for lack of time this determination was not made. It was my
impression, however, that the dull blackish-red stems of the former
contained more anthocyan than the brilliant stems of the latter. If
this impression is correct, the three grades of pigmentation of the buds
are completely associated with three grades of pigmentation in the
stems, but the bud-series and the stem-series run in opposite directions,

' In brackets are given the number of each type which should liave been present if tlie
entire progeny had been grown to maturity. Tliese are the significant numbers, as tlie
proportionality among the observed numbers is distorted by the fact tliat a relatively larger
number of unspotted rosettes were grown to maturity, than of spotted ones.

G. H. Shull 93

thus: green with dark red, pink with bright red, red with gi-een. The
fine red specks often seen on the ovaries show a partially independent
series, being absent in (a), nunierons in (b) and scarce in (c).

The distribution of the adult plants shows that the classification of
the rosettes into spotted and unspotted categories was not in this case
a natural grouping, as it was in the reciprocal rubricalyx-rubrinervis-
crosses, and this difference is doubtless due to the fact that my
0. Lamarckiana has moderately spotted rosettes while 0. rubrinervii
is unspotted, as sho%vn by Plates V and VI (bottom series in each). All
plants of groups (a) and (b) had spotted rosettes, and two-thirds of the
former were noticeably more strongly spotted than the rest of the
spotted plants. All the imspotted rosettes and most of the moderately
spotted ones developed into plants of type (c). It is thus seen that
although the pigmentation of the rosette is not in this hybrid com-
bination, as sharply diagnostic of the natural groups, the nature of the
association between the pigmentation of the rosette and that of the
adult organs is the same, in direction, in the ruhricalyx-Lumurckiana-
cross as in the rubricalyx-rubrineims-cross. Stated more generally,
there is a positive correlation between the red-pigmentation of the
rosette-leaves and that of the adult stems, and a corresponding negative
correlation between the red-pigmentation of the rosette and the red-
pigmentation of the buds.

Oenothera Laiuarckiana x rubricalyx Fi.
Pedigree Nos. 118(10) x 11410(1) = 1235.

Seeds of this cross produced 117 plants which were at one time
grouped provisionally into 36 Lamarckiana-Uke, 18 rubricalyx-like
and 37 rubrinervis-\ike, besides a considerable number of individual
aberrants. As these names proved later to be inappropriate it is
desirable to translate them for our present use into terms of their
pigment-characters. The " Lamurckiana-\ike" group and the " rubri-
cali/x-Vikc " gi-oup had red-spotted rosettes, and the " rubrinervis-Wke "
group had unspotted rosettes. Unfortunately, several of the divergent
rosettes which received individual description, were not noted with
reference to the presence or absence of red spots, and only 113 are now
classifiable on the basis of the red spotting of the rosette-leaves ; of
these, 64 were spotted and 49 unspotted. Most of the slightly aberrant
rosettes developed into adult plants not perceptibly divergent from
other plants which had not been noticeably aberrant in the rosette-

04 Necjative Correlation in Oenothera ffi/briffs

stage. Two had hc^avier buds than the rest, nearly lik(! those of pure
rubriuervis in size and form, but each of these had greenish steins and
n(bricali/:i-co\ored buds ; they are included among the number referred
to group (c) below. Aside from these two plants and several specimens
which failed to bloom, the 78 plants which had been set into the field
were referable to three phenotypes, indistinguishable from those pre-
sented by the reeiprocal family. The several types of j'igmcntation
were distributed in the following proportions:

(a) 7 [9]' with dull, dark-red stems and buds devoid of anthocyan.

{b) 17 [22] with brilliant red stems, pink cones and green hypanthia.

(o) 51 [82] with greenish stems and intensely red hyjjanthia and
cones.

The relation of these groups to the rosette-characters of the .same
faniilv are the same as in the reciprocal family; both (a) and {h) were
completely included among the sjwtted rosettes, while (c) included all
the unspotted rosettes and 26 (i.e. essentially 50 per cent) of the
spotted ones.

Similar phenomena in other Oenotliera-crosses.

The negative correlation between the redness of the stems and that
of the buds, which has been so strikingly manifested in these rubricalyx-
crosses, is by no means limited t(j the combinations of 0. ruhricalyx. In
an extensive series of crosses between 0. Ltnnarckiaita and several
biotypes of 0. cruciata, glimpses of the same inverse relation in the
distribution of the red pigmentation, have been frequently seen. The
results of the latter crosses will be published in detail in another place,
and only several examples of many that might be presented will be
introduced here to illustrate the criss-cross distribution of the red
pigment on the stems and buds. The forms of 0. cruciata have green
buds and usually more or less strongly reddened stems. 0. Lamarc/dana
has pink cones and green hypanthia associated with only moderate
reddening of the stems, the stems having a degree of red-pigmentation
similar to that of the above-described " greenish "-stemmed plants
among the ruhricalij.v-hyhrids. A cross between a certain elementary
form of 0. cruciata. $ and 0. Lamarckiana. </ produced in the F^
(Ped. No. 1140) four types characterized as follows with respect to
pigmentation :

(a) 2 [2]' with red stems and buds rntirely green.
' See footnote on p. 92.

G. H. Shull 95

(6) 10 [18] with bright red stems, very slightly reddened bud-cones
and green hypanthia.

(c) 2 [3] with pink stems, and with cones and hypanthia red
throughout.

(d) 5 [5] with stems nearly green and buds entirely green.

A plant of the type (b), which type made up the bulk of the F,
family, pollinated by a plant of the parental biotype of 0. cruciata
(i.e., a sesqui-reciprocal hybridization of the form {A y. B) x A), produced
43 [97] offspring with red hypanthia and red cones on pink stems, and
5 [6] with faint reddening on the cones, green hypanthia and intensely
red stems (Ped. No. 12107). The parental form of 0. cruciata had only
moderate reddening of the stems, so that the red stems of the hybrid
types {a) and (6) represent a marked intensification of the pigmentation
as compared with both parents, just as the red hypanthia and strongly
red-pigmented bud-cones of hybrid type (c) show a remarkable advance
over the pigmentation of the buds in 0. Lamarchiana, the only one of
the parents which had any red coloration of the buds.

In crosses between 0. LamarckiaMa and two other biotypes of
0. cruciata, the F^ hybrids, when 0. Lamarckiana is the mother, are in
each cross of uniform type, having the bud-cones reddened to about the
same extent as in 0. rubrinervis (i.e., much more strongly reddened
than in 0. Lamarckiana) while all the vegetative parts are pale green
and absolutely devoid of red-pigmentation. The reciprocal crosses
produced in the one case two types, in the other case four types, some
of which had pink cones and green hypanthia, others entirely green
buds, but all had strongly reddened stems, the green-budded plants
having a stronger pigmentation of the stems than the pink-coned plants.
In the latter pedigrees the green-budded forms can be partially sorted
out from the pink-coned forms in the rosette-stage, because of the more
prominent red spots on the dorsal surface of the young rosette-leaves
in the former.

The pigmentation of 0. rubrinervis as compared with its parent
0. Lanuirckiana, may be related to the same phenomenon, for the bud-
cones of the mutant-form are much more strongly pigmented than those
of 0. Lamarckiana, while the rosettes are entirely free from red spots
and the stems are nearly gi-een, while 0. Lamarckiana, on the other
hand, has the rosettes sparsely spotted and the stems moderately
reddened. Thus, 0. rubrinervis shows a progressive variation in the
amount of red pigment in the buds, while the leaves and stems present
a retrogressive variation in respect to red pigmentation.

96 Negative Correlation in Oenothera Hybrids

Discussion.

The behavior of tlic red pigments in these 0. nibricdlyj'-hyhr'uU,
is in striking contrast with that reported by Gates (1909 to 1918 b),
whose reciprocal crosses between 0. rubrical i/.r and 0. Lamarckiaiia
appeared to consist j)redoniinantly of rubricaly.r and Lainarckiana. To
what extent the differences between his results and mine are due to the
very slender basis for the conclusions regarding his crosses of these
species, cannot be determined, and consequently it is impossible to say
how much of these differences is to be referred to putative genotypic
differences between the particular individuals which entered into his
crosses and those which were used in mine. Whatever may be the fact
in regard to the lack of genotypic identity between his parent plants
and mine, the results described in this paper have an important bearing
on the several propositions set foi'th by Gates in regard to the oiigin
and genetic behavior of the rubricuii/.r-chiir.Kiter, namely (<() that the
difference between 0. rubricalyx and 0. rubriaervis is a purely quantita-
tive one ; (6) that the /7(6/"ica7?/./'-character is a typical monohybiid
Mendelian character; and (c) that the method of inheritance of a
character is determined by the nature of that character itself.

Many Mendelian color-patterns have been discovered in various
plants and animals, which are inherited quite independently of the
actual quantities of pigment present in the organism as a whole, th
same pattern being associated sometimes with weak pigmentation at
other times with intense pigmentation ; dissimilar and independently
inheritable patterns may also affect different parts of the same indi-
vidual, and in every such case it is demonstrable that a " qualitative "
and not alone a "quantitative" difference is present. Such experiences
incline the geneticist to the interpretation of any striking change in
a color-pattern as something more than a (juantitative change in the
amount of pigmentati(jn present. Still, in a case as simple as that
which Gates's material seemed to him at first to present, in which the
new color-pattern completely includes the original one, the interpre-
tation of the change as a purely quantitative one is possible, though
perhaps not in any case particularly probable. Gates (1914) has now
shown that in the case of the rubricalyx-ch-ArMiiev, also, the pattern is
measurably independent of the actual quantity of pigment, since he
secured pale red buds with the characteristic /•wicicatyr-distribution of
the pigment. The results from my crosses show even more strikingly

e

G. H. SiiuLL 97

that the mere quantity of anthocyan which a plant produces does not
make the diflference between the color-pattern of 0. ruhricalyx and
that of 0. rubnnervis, for certainly those plants with red-spotted leaves
and dark-red or brilliant-red stems, produced many times as much
anthocyan as the greenish-stemmed plants, though only the latter had
the rubricalyx-type of buds.

It is interesting to see that even Gates (1914) occasionally catches
a glimpse of the fact that the difference between 0. rubrinervis and
0. rubricalyx is something more than a mere quantitative difference in
the amount of red pigment produced by each. He says (1914, p. 246)
■ " It is then perfectly clear that, although extent of pigment on the buds
behaves, with very few exceptions, as a definite unit-character, showing
the phenomena of dominance and absence ; yet the amoimt of pigment
is very probably reduced to a half in the Fi hybrids, and it is certainly
diluted very much (probably one-half) again on crossing back with

grandiflora The cause of the definiteness of distribution of the

pigment in the buds is a problem in morphogenesis." After this
statement one may be surprised to read (p. 270) that " the most careful
observation and study shows that the red pigmentation-character R is
in the last analysis, inherited in quantitative fashion."

The Mendelian behavior of the rubricidyx-ch&rsicier was inferred
by Gates from very insufficient evidence. When an attempt is made
to interpret my results on a Mendelian basis, difficulties are encountered
which seem at present nearly insurmountable. If we suj^pose that my
original ruhricalyx--p\B,\\i (No. 11410(1)) was a heterozygote in respect
to two independent determiners for the rubricalyx-ty^e of buds, the
progeny secured by self-fertilizing this plant was in as close agreement
with expectation, as the small number of plants would require, the
ratio being 10'7 : 1 instead of 15:1; but if there were no further
complications, such a constitution for the ruhricalyx parent would lead
to the expectation of rubricalyx and non-rub ricalyx in the ratio 3:1 in
every cross between this plant and other plants which lacked both of
the supposed determiners for the rnbriculyx-c\\'Ava,cieT. Instead of this,
the ratios in the four hybrid families here reported, were 0"91 : 1, 144 : 1,
1"80 : 1 and 2"65 : 1, or if we combine the two rubricalyx-rubrinervis
families, the result is 165 : 183 or nearly 1 : 1, while the two Lamarckiana
crosses combined give 161:75, or about 2:1. Both of these ratios
actually occur in typical Mendelian inheritance, and could be accounted
for here by such additional assumptions as these ; namely, (a) that my
rubrinervis plant, No. 1123(8), not only lacked the two independent

Journ. of Gen. iv 7

98 Neijatim Cornhition in Oenothera Ili/brUh

rubricalyx-determincvs, but that it was also heterozygous for a thinl
gene which inhibits the action of one of the genes for the rubricahjx-
type of pigmentation, but not the other; and (6) that my Lamarcfciaiia-
plant. No. 118(10), contained a factt)r whose union with one of the
rubricali/x-determineTS produces, in the absence of the other, a nun-viable
zygote. While such assumptions are perfectly proix-r as working
hypotheses, they have no other value. Assumptions of a still more
radical kind would have to be made to account for the peculiar
alternative relation between the pigmentation of leaves and stems on
the one hand and that of the buds on the other hand.

In the enormous mass of genetic data already recorded for the
Oenotheras, there is but here and there a situation which bears more
than a remote resemblance to a Mcndelian behavior, and in these
cases the observed phenomena usually present only a more or less
misshapen caricature of the beautiful regularity of procedure which has
such far-reaching applicability among many other groups of organisms.
It appears to me undesirable therefore to speak of the rubricali/x-
character as a Mendelian unit-character because it happened to
constitute 75 per cent of one flimily of 44 plants. I believe that the
only other character in Oenotheras which has been accepted as Mendelian
in inheritance, namely, the brevinti/lis-chareicteT, may well be put to the
test of a fuller genetic analysis.

In view of these facts, one can only view with astonishment the
performance of Heribert-Nilsson (1912) in maintaining that the remark-
able series of genetic puzzles presented by the Oenotheras can find an
explanation through the recombinations of plural Mendelian determiners.
His entire thesis falls to the ground the instant we begin to figure out
some of the very simplest and most obvious consequences of such an
explanation. His abandon in the application of this hypothesis was
made possible only by his belief that students of the Oenotheras have
generally failed to use strictly individual analysis in their investigations.
Having myself never mixed the seeds from two different mothers or
from two different crosises, I am unwilling to believe that Heribert-
Nilsson has not greatly overestimated this source of diflBculty in
interpreting the genetic phenomena in Oenothera, although it is a
valuable service to have pointed out so strongly as he has done the
importance of the strictest possible adherence to the "isolation principle."
The importance of this emphasis may be seen, when so careful a worker
as Dr. Gates grows a culture from the mixed seed of four different self-
fertilized parents, belonging to a group in which there was obvious

G. H. SiiuLL 99

genotypic impurity, on the ground that " this made no difference in the
experiments [he] then had in view" (Gates, 1913b, p. 143). It is a
little difficult at the present time to imagine the nature of those genetic
experiments which would not be injuriously affected by such an origin
of the foundation stock. It can only be a matter for regret that Gates
(1914) has also used for his so-called "reciprocal" crosses between
0. rubricalyx and 0. grandifiora, a strain of the latter species from
Alabama, for the one cross, and one from Birkenhead, England, for the
" reciprocal " cross, especially in view of the fact that he has found
much evidence of complex hybridizations in the latter locality (Gates,
1913a). Finally, his "back-crosses" of the F^ plants from this second
hybrid family have been made not upon plants of the parental strain,
but upon those of the Alabama strain. It is hardly to be hoped that
the elements of genetic behavior in Oenothera will be discovered by
these methods.

The conclusion reached by Gates (1910) that the method of
inheritance of a character is determined by the nature of the character
itself, is also brought sharply into question by the peculiar situation
in my 0. ruhricalyiV-cmsses, for I am clearly dealing with the same
character which Gates has repeatedly called a monohybrid Mendelian
dominant ; but in my cultures it is either not Mendelian at all, or, if
Mendelian, is affected in a complex way by several different determiners.
The remarkable diversity in the nature of the characters which have
been proved to be typically Mendelian in inheritance in various plants
and animals, should have made Gates's conclusion impossible. It is not
the externally visible, physical or chemical nature of a character which
determines the method of its inheritance, but the nature of the
inheriting-" mechanism " to which it is related, and the manner in
which its determiner or determiners are related to that mechanism.
Baur (1910) has discovered a case which clearly illustrates this point,
and I also have been able to confirm his results (ShuU, 1914). We
have shown that " chloralbinism " in plants may result from several
different causes. In some cases the absence of chlorophyll is due to
the absence of a definite Mendelian gene, inherited equally well through
both the male and female gametes; in other cases it appears to be a
purely cytoplasmic defect, inherited in characteristically non-Mendelian
ways, sometimes only through the mother, sometimes through the
father and the mother, but with such ii-regularity that its inheritance
cannot be related to Mendelian phenomena. Whether Mendelian or
non-Mendelian, the character itself appears to be the same, namely, the

100 Neqatwe Correlation hi Oenothera ITjihruh

absence of the chlurojjlasts. Numerous investigators have sliown tliut
the anthocyan pigments of many plants are determined in (juantity,
quality and distribution by normal Mendelian genes, but the studies
described in this paper, as well as those of Gates, strongly indicate that
in Oenothera the inheritance of the red-pigmentation is determined by
some other hereditary system, and the same inference may be drawn
from most of the other genetic phenomena thus far recorded for Oeno-
theia. Further experimentation must discover a mechanism adecpiate
for the interpretation of these genetic phenomena. Until that mechanism
is found it will be impossible to decide what constitutes a unit-character
in Oenothera, or to decide whether any paitieular genetic ditf'eieiitiation
represents a case of segregation, " fractionation," or some other method
of distribution (jf characters.

Summary.

An investigation of the genetic phenomena presented by Oenothera
ruhricaly.T Gates and its hybrids has shown that the bright red
hypanthia and cones of that .species are separable in inheritance from
the brilliant red stems with which, according to Gates's description, it
was always associated in his cultures.

In the Fi hybrids from reciprocal crosses between this species and
0. ruhrinervis and 0. Lainarckiana a remarkable series of negative
correlations appear in the distribution of the red pigment, the brilliantlv
pigmented buds characteristic of 0. ruhrlady.r being invariably associated
with a low degi'ee of red-pigmentation in the stems and rosettes ; pink-
coned buds with green hypanthia, characteristic of 0. Lamarckiaiin,
being on the other hand, invariably associated with brilliant red stems,
while buds entirely free from anthocyan are associated with dull dark-
red stems.

A self-fertilized plant of 0. ridmcalyx produced offspring having
rubrical yx-hwds and green buds in the ratio 10'7 : 1, all the green-
budded plants having Jitme^^a-stature and chai-acteristic dark-red stems.
One plant having r)/7wica?//;t-pigmentation was likewise of the dwarf

typf-

The ratio of ruhriculyx-hwAAeA plants to non-rubricalyx in the
crosses with nibrinervis was approximately 1:1, and in crosses with
Lamarckiana 2:1.

By complicated auxiliaiy hypotheses these ratios could be explained
in accord with Mendelian inheritance, but the fjxct that Oenothera
apparently has a unique mechanism for the distribution of hereditary

JOURNAL OF GENETICS, VOL. IV. NO. 1

PLATE V

JOURNAL OF GENETICS, VOL. IV. NO. 1

PLATE VI

a H. Shull 101

characters makes such subsidiary hypotheses of no value except as a
basis for further investigations.

Other Oenothera crosses are cited, which indicate that the inverse
relation between the red-pigmentation of the buds and that of the
stems is not limited to crosses of 0. ruhricalyx. Oenothera ruhrinervis
represents a progressive variation in the pigmentation of the bud-cones
and a retrogressive variation in the pigmentation of the stems.

It is held that three conclusions arrived at by Gates regarding the
origin and genetic nature of the ri(6ricai;(/*'-character are erroneous,
namely, (a) that the character represents a purely quantitative differ-
ence from 0. ruhrinervis, (b) that it differs from the latter species in
a single monohybrid Mendelian unit, and (c) that the nature of a
character itself instead of the nature of the inheriting-mechanism to
which it is related, determines the manner of inheritance of that
character.

LEGENDS FOR PLATES.

PLATE V.

Bosettes of 0. ruhricalyx (upper series), 0. riibrinenus (lower series) and of their
reciprocal Fi hybrids. Each set of 3 hybrid rosettes stands next to those of the
seed-parent type. Inside diameter of pots about 7'5 cm. Photographed April 19, 1913,
about 11 weeks after the seeds were sown.

PLATE VL

Eosettes of 0. ruhricalyx (upper series), 0. Lamarckiana (lower series) and of their
reciprocal Fj hybrids. Arrangement the same as in Plate V. Photographed April 9,
1913, about 10 weeks after the seeds were sown.

LITERATURE CITED.

Baur, E. 1910. "Untersuchungen iilier die Vererbuiig von Chromatophoren-
merkmalen bei Melaudrium, Antirrhinum und Aquilegia. Zeitschr. f. ind.
Abstamm. u. Vererh. Bd. iv. pp. 81 — 102.

Heribert-Nilsson, N. 1912. "Die Variabilitat der Oenothera Lamarckiana und

das Problem der Mutation." Zeitschr. f. ind. Abstamm. u. Vererh. Bd. viii.

pp. 89—231.
Gates, R. R. 1909. " An Analytical Key to .some of the Segregates of Oenothera."

Ann. Rep. Missouri Bot. Gard. Vol. XX. pp. 123 — 137. See p. 133.
. 1910. "The Material Basis of Mendehan Phenomena." Amer. Nat. Vol.

XLiv. pp. 203—213.

7—3

lOii Negative Correlation in Oenothera Hyhrith

Gates, R. E. 1911. " Studies ou the Variability and IleritaUility of Pigmentation
in Oenothera." Zeitschr. f. itid. Abstamm. u. Vererb. Bd. iv. pp. 337 — 372.

2 figs, of 0. ruhricalyx and 1 colored plate showing contrast between buds of

0. ruhricalyx and 0. rubrinern.i.
. 1912. "Mutations in Plants." Bot. Jour. Vol. ii. pp. 84 — 87. 1 fig. of

0. rubricalyx.
. 1913a. "A Contribution to a Knowledge of the Mutating Oenotheras."

Trann. Limiean Soc. London, Botani/, Vol. vin. pji. 1— (iT. C pis., including

6 figs, of 0. ruhricalyx.
. 1913b. "Tetraploid Mutants and (.'hroniosonie Mechanisms." Biul. Centralbl.

Bd. xxxiu. pp. 92—150.
. 1914. " Breeding Esperinients which show that Hybridization and Mutation

are Independent Phenomena." Zeitschr. f. ind. Ah.itanim. u. Ventrb. Bd. xi.

pp. 209—279. 3 figs, of 0. ruhricalyx.
Shhll, G. H. 1914. "tjber die Vererbung dor Blattfarbo bei Melandrium."

Ber. d. Deutsch. Bot. Gese/L Bd. xxxi. Generalversaramlungs-Heft, pp. 40 — 80.

ON A SUPPOSED SYNTHESIS OF ANTHOCYANIN.

By M. WHELDALE,

Fellow of Newnham College, Cambridge ;

AND H. Ll. BASSETT,
Tnnity Hall, Cambridge.

{From the Laboratory of the John lanes HoHicidtural Institution,

Merton, Surrey, and the Balfour Laboratory, Cambridge.)

In a recent paper dealing with the anthocyanin pigments of plants
Everest' makes suggestions which are quite in contradiction to the
views generally held on the subject.

His main contention is that anthocyanins are reduced products of
flavones, whereas most investigators have considered them to be oxidised
products of some aromatic chromogen. He also raises the question of
the mechanism of their production and the first part of his paper is
devoted to a criticism of an hypothesis which was brought forward b}'
one of us some years ago.

The said hypothesis was based upon various observations comiected
with the distribution, appearance and disappearance of anthocyanin
pigments in plants, whereby it appeared that the formation of the
latter is in some way intimately connected with photosjaithesis and
the products of this process, i.e. sugars. Hence it was suggested ten-
tatively", as there was no absolute evidence either for or against it, that
if anthocyanins are formed from flavones (which occur in the plant as

' Everest, A. E., " The Production of Anthocyanins and Anthocyanidius," Roy. Soc.
Proc. 1914, B, Vol. lxx;cvii. p. 444.

- Wheldale, M., " On the Formation of Anthocyanin," Jourii. of Genetics, 1911, Vol. i.
p. 133. And later, Wheldale, M., "The Flower Pigments of ^7i(in7iinumma/«s. I. Method
of Preparation," Biochemical Joiirn. 1913, Vol, vii. p. 87.

104 On a Supposed Synthesis of Anthoqjanin

glucosides), the reactions controlling the formation of anthocyanin might
be as follows :

glucoside (of tlavone) + water ±^ chromogt'n (tlavime) -/- /2-tx^,<*^
"Frrrrr^r y ( flavone) + oxygen = anthocyanin'.

It was suggested that one or more hydroxyls, though not necessarilij
all, of the flavones were combined with sugar and that after hydrolysis
of certain, though again not all, of the hydroxyls, further changes, such
as oxidation or condensation, or both, might take place at these points
thus opened to attack, with the formation of anthocyanin. Hence the
anthocyanins themselves would be glucosides, and at no time would the
products taking part in the reaction be completely in the nonglucosidal
state.

The first criticism offered by Everest is that there are no known
glucosides of the flavones having more than one or two hydroxyl gi-oups
substituted by sugar molecules. The hypothesis was brought forward
in full knowledge of this fact, but it must be borne in mind that no
accurate work has been done with a view to actually ascertaining the
number of sugar molecules attached to flavones in the plant. Certain
stable glucosides of various flavones, with only one or two sugar mole-
cides attached, have, from time to time, been extracted and identified,
and probably the fact that they could be identified is due to their
stability. It is quite conceivable that, gi'eater precautions being taken,
other and less stable compounds might be isolated. No special pre-
cautions have in fact been taken, certainly in the majority of cases, to
avoid decomposition of possible unstable glucosides. Until further
investigations have been carried out on this point, no definite statement
can be made as to the number of sugar molecules attached to flavones
while in the plant.

The second criticism offered by Everest is based upon a misconception
of the hypothesis. According to the evidence of Willstatter and Everest'',
anthocyanins (as glucosides), can be distinguished from anthocyanidins
(non-glucosides), by their solubility in amyl alcohol, the anthocyanidins
being soluble.

Hence if the flavones are completely hydrolysed and then give rise
to non-glucosidal anthocyanin (which is an incorrect interpretation of
Wheldale's hypothesis) it should be possible to isolate anthocyanidins

1 The second equation is incorrectly quoted in Everest's paper, no allowance being made
for condensation.

2 Everest, loc. cit.

M. Wheldale and H. Ll. Bassett 105

from the plant by means of amyl alcohol and this, as Everest notes, is
contrary to all observation.

But, as stated above, it is not suggested in Wheldale's hypothesis
tliat anthocyanins or Havones are at any time entirely free from sugar
so that the hj-pothesis is not, on this point, in contradiction to observed
facts.

Further, still on the incorrect interpretation of the hypothesis,
Everest states, " one would expect that by taking the yellow glucoside,
hydrolysing, then reducing with removal of sugars, the anthocyanidin
produced would combine with the sugar present to form an anthocyanin.
This is not the case." Under any circumstances this criticism would
be of no value as it is well known that glucosides are not formed in
vitro, except in a few cases under very special conditions such as are
certainly not realised in the above experiment.

There is however yet another point, namely that Everest maintains
that no hydrolysis at all of flavone glucosides is necessary as a pre-
liminary to the formation of anthocyanin (glucosidal). For he obtains
anthocyanin from extracts of many plants containing flavones as gluco-
sides by simple reduction in the cold (though this result was not
obtained with artificial quercitrin, a glucoside of quercetin). The value
of this criticism depends on whether the products formed by reduction
are really anthocyanins.

This question must now be considered, and forms the second main
part of Everest's paper. The reactions described are as follows. When
the flavones, quercetin, morin and luteolin are treated, in alcoholic
solution acidified by hydrochloric acid, with nascent hydrogen formed,
for instance, by the action of sodium amalgam, a purple-red or red
colour is produced. In the case of quercetin (we do not know whether
this is equally true for morin and luteolin) the red product gives with
alkalies a gi-een reaction similar to that given by anthocyanin.

Everest has prepared these red substances from quercetin and from
various plant extracts. When quercetin was used t~he red product was
soluble in amyl alcohol — hence it was regarded as an anthocyanidin.
When plant extracts were employed, the red product was insoluble in
amyl alcohol — hence it was considered to be an anthocyanin.

Everest apparently assumes the red products to be anthocyanins or
anthocyanidins, as the case may be, solely on the basis of the gi'een
reaction with alkalies, since the mere fact of the solubility or insolubility
of the substances in amyl alcohol cannot be taken as evidence of their
anthocyanin nature.

106 On a Supposed Sf/nt/iem's of Anthocyanin

Moreover the fact that both natural aiitliocyaniii and the artificial
red products can be reduced to a colourless form and then reoxidised to
the coloured state, cannot be taken as proving that the artificial sub-
stance is anthocyanin, since innumerable coloured aromatic substances
give leucoconipounds on reduction which regain their colour on oxidation.

The grounds for thinking tliat these substances are anthocyanins
seem to us slight, and our observations lead us, in the absence of further
evidence, to feel considerable doubt on the point for the following
reasons :

1. The rod product from (juercetin is soluble in ether whereas all
natural anthocyanins are quite insoluble in this solvent.

2. The only case in which anthocyanins and their antecedent
chromogen have been analysed is that of Antirrhinum, and here the
anthocyanins are oxidised, and not reduced products, of the flavone
concerned, i.e. apigenin'.

3. When apigenin is treated in alcoholic solution with sodium
amalgam and acid, as was done with quercetin, we obtained a red
product and the reaction clearly followed a similar course in the two
cases. Analyses showed the product from apigenin to be a reduced
substance and its reactions with acids and alkalies do not in any way
resemble those of anthocyanin. With alkalies the red colour is deepened
and with acids the red colour becomes yellowish.

Since, in the case of apigenin, the product is certainly not an antho-
cyanin, it obviously throws doubt on Everest's assumption that the
similar products from quercetin and other flavones are anthocyanins.
Thus, in the only case where the flavone and the naturally derived
anthocyanin have been isolated, the product formed by reduction of the
same flavone is quite a different substance from the natural anthocyanin.

The evidence of a considerable amount of work by Keeble, Armstrong
and Jones^ shows that in the vegetative organs as well as in the flowers
of various plants the distribution of anthocyanin coincides with the

' Wheldale, M. and Basset, H. LI., "The Flower PignientB of Antirrliiniim niujiis.
3. The Red and Magenta Pigments," Biochemical Juuiii., 1913, Vol. vii. p. 87.

- Keeble, F. and Ainistionp, E. F., " The Distribution of Oxydases in Plants and their
Role in the Formation of Pigments," Roy. Soc. Proc, 1912, B, Vol. i.xxxv. p. 214 : " The
Role of Oxodases in the Formation of the Anthocyan Pigments of Plants," Journal of
Genetics, 1912, Vol. ii. p. 277: Keeble, F., Armstrong, E. F., and Jones, W. N , "The
Formation of the Anthocyan Pigments of Plants. Part 4. The Chromogons," Hoy.
Soc. Proc, 1913, B, Vol. i.xxxvi. p. 308: "The Formation of Anthocyan Pigments
of Plants," Part G, Roy. Hoc. Proc, 11113, B, Vol. i.xxxvii. p. 113.

M. Wheldale and H. Ll. Bassett 107

presence of oxidising enzymes. Anthocyanin is therefore formed in
tissues in which oxidation most readily takes place. The above authors
have latterly inclined to the view that reduction precedes oxidation, but
this view rests on the supposition that the products of reduction of the
flavones are anthocyanins.

It is difficult to reconcile Everest's assumption that anthocyanins
are reduced flavones, with the fact that they are shown to occur only
in those parts of plants where oxidases, and therefore oxidations, occur.

There remains then the question of the derivative from quercetin
which gives a gi'een alkali reaction. This substance was prepared by
us from an alcoholic solution of quercetin ; the residue, after evaporation,
was extracted with ether containing a little alcohol and several com-
bustions were made of the product. The results showed the product to
be a reduced derivative of quercetin.

A determination of the molecular weight by the raising of the
boiling point of alcohol gave a molecular weight approximately equal
to that of quercetin. Hence the substance is a simple reduction
product of quercetin, whereas the natural anthocyanins derived from
apigenin are both oxidised and condensed products. Nothing can be
gained at present by comparing the composition of the reduction
product from quercetin with other analyses of natural anthocyanins,
since the antecedent chromogens of the latter are not known.

Further discussion on the matter is useless in the absence of exact
experimental evidence. In the case of quercetin, in order to secure any
convincing evidence, it would be necessary to isolate a natural antho-
cyanin known to be derived from quercetin and to compare the results
of its analysis with those obtained for the reduction product.

HEREDITY AND MEMORY

The Henry Sidgwick Memorial Lecture

delivered at Newnham College, Cambridge
9 November 1912

By JAMES WARD, Sc.D, Professor of Mental Philosophy in the University
of Cambridge, Author of The Realm of Ends or Pluralism and Theism.
Crown 8vo. Paper covers. Is. net. Cloth, Is, 6d. net.

" Professor James Ward, in bis clioice of Heredity and Memory as the subject of tlie Henry
Sidgwick Memorial Lecture in 1912, has earned the thanks of all who are interested in the
important and highly controversial problems which the topic inevitably suggests. The lecture
was without question a serious and weighty contribution to the growing volume of dissentient
criticism of Weismann's conception of heredity." — British Medical Journal

MENDEL'S PRINCIPLES
OF HEREDITY

By W. BATESON, M.A., F.R.S., V.M.H., Director of the John Innes
Horticultural Institution. Third impression with additions. With
3 portraits, 6 coloured plates, and 38 other illustrations. Royal 8vo.
125. net.

In the past three years the progress of Mendelian analysis has been very
rapid, and the author has endeavoured in a series of brief Appendixes to
acquaint the reader with the nature of the principal advances made.

' ' This third edition shows how rapid has been the progress of Mendelian analysis. It deals
with heredity of colour, heredity and sex, gametic coupling and spurious allomorphism, double
flowers, evidence as to Mendelian inheritance in man, biological conceptions in the light of
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The work is beautifully illustrated." — Chicago Medical Journal

"A new impression cannot faU to be welcomed. ...D/ende/'s Principles of Heredity/ is already
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The various waves of biological thought are constantly intersecting, mingling, and passing on
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THE METHODS AND SCOPE
OF GENETICS

By W. BATESON, M.A., F.R.S., V.M.H.

Crown Svo. Is. 6d. net.

"Professor Bateson tells how Mendel's law works out with the colours of certain flowers,
moths, and canaries, and with colour-blindness in men and women. More than this, he describes
the outlook over this field of research in a manner that will greatly interest and attract all in-
telligent people, for, as he rightly says, ' Mendel's clue has shown the way into a realm of nature
which for surprising novelty and adventure is hardly to be excelled.' " — Morning Post

Cambridge University Press, Fetter Lane, London
C. F. Clay, Manager

CONTENTS

AU Rights reserved

PAGE

L. DoNCASTER. On the Relations between Chromosomes, Sex-limited

Transmission and Sex-determination in Abraxas grossulariata.

(With Plates I— III) 1

R. C. PuNNETT and P. G. Bailey. On Inheritance of Weight in

Poultry. (With Plate IV and 3 Text Figures) .... 23
H. M. Leake. A Preliminary Note on the Factors controlling the

Ginning Percent of Indian Cottons . . . . . . ^ 41

N. I. Vavilov. Immunity to Fungous Diseases as a Physiological

Test in Genetics and Systematics, exemplified in Cereals . . 49
H. E. Jordan. Hereditary Lefthandedness, with a Note on Twinning.

(Study III.) (With 80 Figures) 67

George Harrison Shull. A Peculiar Negative Correlation in

Oenothera Hybrids. (With Plates V and VI and 1 Text Figure) S3
M. Wheldale and H. Ll. Bassett. On a Supposed Synthesis of

Anthocyanin .^ . . . . 103

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Artificial Parthenogenesis and Fertilization. By Jacques
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This new work presents the first complete treatment of the subject of artificial
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the bearing of the facts recorded and discussed in the book goes beyond the special
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British Medical Journal. The subject of the book is an analysis of the mechanism by
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Heredity and Eugenics. B}' John M. Coulter, William E.
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presentation of the subject for readers untrained in biology as well as for students.

Contents : I. Recent Developments in Heredity and Evolution : General Introduction.

II. The Physical Basis of Heredity and Evolution from the Cytological Standpoint (John
Merle Coulter, Professor and Head of the Department of Botany, the University of Chicago).

III. The Method of Evolution. IV. Heredity and Sex (William Ernest Castle, Professor
of Zoology, Harvard University). V. Inheritance in Higher Plants. VI. The Application
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Relation to Eugenics (Charles Benedict Davenport, Station for Experimental Evolution,
Carnegie Institution of Washington).

British Medical Journal. Those who are desirous of arriving at an estimate of the
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"Heredity and Eugenics."

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most of the lectures there is an admirable reserve, not to say skepticism, in the treatment
of large questions which the public is often misled to regard as already and finally settled.

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Volume IV OCTOBER, 1914 No. 2

OUR PRESENT KNOWLEDGE OF THE CHEMISTRY

OF THE MENDELIAN FACTORS FOR FLOWER- .„,«ar^
COLOUR. ■ ^^' ^«**

rt.M aNICM.
By M. WHELDALE, oakDBC*,

Fellow of Newnham College, Cambridge, and formerly Research Student
of the John Innes Horticultural Institution, Merton, Surrey.

Since the inheritance of flower-colour involves, in the majority of
cases, the inheritance of some kind of anthocyanin pigment, any know-
ledge of the chemistry of the factors for colour must of necessity be
concerned with the chemistry of anthocyanin. During the last few
years a considerable amount of work has appeared, i.e. that published
by Combes, Grafe, Tswett and Willstatter, on the chemistry of antho-
cyanin apart from its connection with Mendelian problems. It seems
advisable at every stage to bring the results of such work to bear, when
f)ossible, upon the evidence obtained by those who have approached the
subject more directly from the Mendelian point of view.

In the present paper an attempt has been made to state, as far as
possible, from all the evidence available, exactly how much we know of
the chemical mechanism underlying the Mendelian factors for flower-
colour.

The colour varieties of Antirrhinum majus having provided useful
material for the chemical interpretation of some Mendelian factors, an
account will be first given of the pigments of this species.

The Varieties of Antirrhinum majus.

The original wild type of Antirrhinmn majus has magenta flowers,
the colour being due to at least seven pairs of factors (Wheldale, 2-5, 26,
29 ; Baur, 5, 6), but of these, only four need be mentioned in the present
paper, and the type homozygous in all factors may be represented as :

YYIIRRBB magenta.

Joum. of Gen. ly 8

110 Cliemistnj of MendeUan Factors for Flower-Colour

Of these factors :

Y represents the power to produce a very pale yellow (ivory) pigment
in the tube of the corolla and a deep j'ellow pigment in the lips of
the corolla with a still deeper yellow patch' on the palate (PI. V^II,
fig. 2). The composition of a plant bearing yellow Howers may be
represented as:

YY{y)nrrB(b)B(b) yellow.

/ represents the power to form ivory pigment in the tube and lips, and
to inhibit the formation of yellow pigment in the lips except on the
palate. The composition of a plant bearing ivory flowers (PI. VII,
fig. 3) is:

YY(ij)II{i)rrB(h)B{h) ivory.

R represents the power to produce red anthocyanin pigment in the
corolla'^. If the ivory factor is absent, the red is mixed with yellow
and the result is bronze (PL VII, fig. 4) :

YY{y)iiRR{r)bh bronze.

If the ivory factor is present, the result is rose dore (PI. VII, fig. 5):

YY{y)II{{)RR{r)hh rose dor^.

B represents the power to convert the red anthocyanin into bluish-red
or magenta anthocyanin. If the / fiictor is absent, the result is a
mixture of magenta and yellow, i.e. crimson (PL VII, fig. C) :

YY{y)iiRR{r)BB{h) crimson,

but if the ivory factor is present, the result is magenta (PL VII, fig. 7) :

YY{y)II{i)RR{T) BB{b) magenta.

If the Y fJictor is absent, the plant produces dead white flowers (PL VII,
fig. 1), though it may be carrying any or all of the remaining factors:

yyI(i)I(i)R{r)R{r)B{b)B{b) white.

1 The patch on tliu palate is common to all varieties except white.

^ In the original publications on Antirrhinum, the factor R was used to denote a
tingeing only of anthocj-anin, an additional factor D being present in the fully coloured
varieties, but this distinction is not really necessary in the present paper.

JOURNAL OF GENETICS, VOL. IV, NO. 2

PLATE VII

1. White

w

2. Yellow

4

•/

r\j

/

/

/

/

r

3. TVORY

■~^\

^.•

4. Bronze

6. Crimson

5. Rose dork

7. Magenta

M. Wheldalb

111

The Yelloiu Pigments of Antirrhinum.

Examination was first made of the yellow pigments of Antirrhinum
and they were found to be members of a group of natural yellow
colouring matters known as flavones. These substances are derivatives
of /S-phenylbenzo-7-pyrone :

and they differ from each other, as regards constitution, in the number
and position of their hydroxyl gi'oups. The accompanying table (on
page 112) sets forth the reactions and distribution of the more common
flavones. The actual extent of their distribution, however, is by no
means represented by the genera mentioned, for our knowledge of the
occurrence of the flavones in plants is practically limited to the researches
of Perkin (21), and a few other workers who have investigated these
particular species solely from the point of view of the dyeing properties
of the plant extracts. The results of these investigations have shown
that the capacity for dyeing in the above cases is due to the presence
of particular flavones.

There is little doubt that the flavones are universally distributed
among plants and form a class comparable to sugars, proteins, tannins,
etc., but no work has yet been done with a view to ascertaining the
distribution of the various members of the class.

The flavones occur as a rule in the plant in the form of glucosides,
one or more of the hydroxyl groups being replaced by sugar, as for
instance :

HO

OC,H„0,

HO CO

In this condition the flavones are very little coloured (see below)
and readily soluble and as such they occur in the cell-sap throughout
the plant. A characteristic reaction of the flavones is to give an

8-2

112 Chemistrji of Mendelian Factors for Flower-Colour

Flavone

Constitutional Formula

Melting Point

HO CO

Products of

Dec(uui)Osition

from Alkali Melt

Pliloroglucin

and
protocateohuio

acid

Distribution

Free and aK various kIuco-
sides in Qiicrcits (bark),
RhiniDui.i (berries), flowers
of Ch('irtinthu.'<, Cratae-
fjus, Viohi^ Prnnii.<, Hibis-
cus, leaves of Ailantkiis,
Rhus,Arctoiita]>hylos, Cal-
luna. Eucalyptus and
many others

Myricetin

HO CO

357° Pliloroglucin Myrica (bark), leaves of

and lihuji, Ilannatoxylon,

gallic acid Aictostnphylos

Fisetin

Above 300'^'

Resoroinol

and

protocatechuic

acid

Iiliu.i (wood)

Chrysin

110

HO CO

>

275° Phloroglucin ropulus (buds)

and
benzoic acid

Apigenin o

HO

HO CO

347° Phloroglucin haavea oS Apiiim, Reseilit

and
p-oxybenzoic
acid

Luteolin . O

HO

HO CO

OH
OH

>

327" Phloroglucin Leaves of Reseda, Genista,

and Digitalis

protocatechuic
acid

Kampherol O

HO

HO CO

27fi°

Phloroglucin

and
p-oxybenzoic

acid

In flowers of Primus, Del-
phitiiuin, leaves of Poly-
gonum, Indigofera, Ro-
bin i a

M. Wheldale 113

intense yellow colour with alkalies ; this reaction is readily seen when
parts of plants, which are free from chlorophyll, are immersed in
ammonia vapour. White flowers under these conditions turn canary-
yellow. When water extracts of glucosides of the flavones are hydrolysed
by boiling with an acid, the flavone separates out from the solution
owing to the fact that it is less soluble than it was when in the form
of a glucoside. The flavones themselves are yellow crystalline substances,
as a rule readily soluble in alcohol, sparingly soluble in ether, and almost
insoluble in water. Their solutions give yellow or orange precipitates
of lead salts with lead acetate and generally a green or brown coloration
with ferric salts. The colour of the flavones is due to the chromophore
group :

o

c

II
o

in conjunction with hydroxyl groups, the auxochromes, the intensity of
colour depending on the position of the latter (Smiles, 22). When the
hydroxyl groups are replaced by sugar or by acetyl or benzoyl radicals
a coloui'less, or practically colourless, compound is produced, since the
effect of the auxochromes is eliminated.

The fact that the flowers of the ivory variety of Antirrhinum
become yellow in ammonia vapour suggested that the pale yellow or
ivory pigment might be a flavone.

Ivory pigment was prepared from the upper lips of ivory flowers in
the following way (W^heldale, 32). Large quantities of material were
boiled with water, filtered, and poured into lead acetate solution which
precipitates the pigment as a canary-yellow lead salt. This was filtered
otf and decomposed by dilute sulphuric acid which precipitates the lead
as lead sulphate, the pigment being set free again in solution. After
filtering off the lead sulphate, the pigment in the dil ite acid solution
was boiled, by which means the glucoside is hydi'olysed, and on cooling,
the free pigment is deposited as a brownish-yellow precipitate which is
filtered off and dried.

The crude pigment was then purified by extracting in a Soxhlet
thimble over boiling ether in which the ivory pigment is soluble and

114 Chemistry of Memlelian Factors Jor Floiver-Colonr

was thereb}' purified from oxidised products formed in hydrolysis. The
ether extract was crystallised from alcohol, the crystals being in the
form of pale yellow plates. The pigment was next converted into its
acetyl derivative by boiling with acetic anhydride. The acetyl deriva-
tive is pure white (owing to replacement of the hydroxyl groups by the
acetyl residue) and crystallises in long needles. The pigment may be
again obtained in a completely pure state by hydrolysing the acetyl
derivative with alcoholic soda solution. From the analyses and melting
points of the acetyl derivative and of the pure pigment (Wheldale and
Bassett, 33), the latter was identified with apigenin, a fiavone of known
constitution occurring in Parsley (Apium Petroselinum), and in small
quantities in Reseda luteula :

O

HO

OH

HO CO

The yellow pigment of the yellow variety was found to be present
in the epidermis only of the lips of the corolla, the inner tissues con-
taining apigenin. Hence the crude pigment, prepared in the way
described above from the upper lips of the yellow variety, always
consisted of a mixture of apigenin and yellow pigment. From pre-
liminary experiments it seemed likely that the yellow pigment was the
flavone, luteolin. On this assumption, a separation of the two pigments
was brought about by means of hydrobromic acid which forms a com-
pound with luteolin but not with apigenin. The yellow pigment was
identified with luteolin by means of its melting point and other
properties and the melting point' of its benzol sulphonyl derivative
(Wheldale and Bassett, 34). Luteolin has been isolated from Reseda
luteolu and it is also found in Genista tinctoria and in leaves of Digitalis.
It is represented as :

P ,OH

HO

OH

CH

HO CO
and owes its intense colour to the ortho-position of the two hydroxyl
groups in the side ring, which structui-e does not occur in apigemn.

M. Wheldale 115

Hence we may regard the dominant ivory factor in Antirrhinum as
the power to inhibit the formation of hiteolin.

The difference between the ivory and 3'ellow varieties can however
be expressed in a more fundamental way. When the flavones are fused
at a high temperature with caustic alkali, a splitting of the molecule
takes place into a phenol, usually phlorogiucin, and a hydroxybenzoic
acid. It is most probable that the flavones, conversely, are synthesised
in the plant from the same phenols and acids, or their derivatives.
Hence the particular flavone synthesised depends on the hydro.Kybenzoic
acid formed in the plant. In the ivory variety the constitution of the
living molecule is such that only a monohydroxy acid can be formed,
whereas in the yellow a dihydroxy acid is formed in addition. More
than this cannot at present be said and no particular benefit is gained
at present by postulating an enzyme connected with the process.

From the white variety no flavone could be extracted, nor do the
flowers give the canary -yellow colour when subjected to ammonia
vapour.

The Anthocijanin Figments of Antirrhinum and Contaurea.

By crossing yellow and ivory varieties with whites carrying suitable
factors, the anthocj'anin-containing varieties can be produced in F^ :

■ YYiirrbb (yellow) x yijURRhh (white) — ■*■ YyiiRrhh (orange)
„ X yyiiRRBB (white) — >■ YyiiRrBh (crimson)

YYIIrrbb (ivory) x yyiiRRbb (white) — ^ YyliRrbb (rose dore)
„ X yyiiRRBB (white) — >■ YyliRrBb (magenta)

Thus it is obvious that in each of the above cases the constituents
of anthocyanin are separated in the two parents but when they come
together again in F^, the pigment can be formed.

The universal distribution of flavones, the similarity in properties
between flavones and anthocyanins, and their intimate connection in
the above crosses led to the suggestion (Wheldale, 27, 28, 30), that
anthocyanins may be derivatives from flavones and that the white
variety contains some factor which modifies the flavone with formation
of anthocyanin.

In the plant, the anthocyanins are present as glucosides, in solution
in the cell-sap of the epidermis of the corolla. From water or alcohol
solutions, they are precipitated as green lead salts, by addition of leac
acetate.

116 Chemist ri/ of Mendelian Factors for Floicer -Colour

Pure anthocyanin was prepared from five varieties of Antirrhinum
in the following way (Wheldale and Bassett, 35). Crude pigment was
obtained by the method described for yellow and iv(jry varieties. Since
anthocyanin only occurs in the epidermis, while apigenin is found in
the inner tissues of the corolla, all crude pigment consisted of mixtures
of anthocyanin and apigenin, and, in some cases, of luteolin in addition.
Anthocyanin was purified from flavones by extracting the mixture for
several months with boiling ether. The residue of crude anthocyanin
was then dissolved in the mininuim amount of alcohol and precipitated
by ether in which it is insoluble. The dried precipitate was again
extracted with ether to remove all traces of flavone. In this way
anthocyanin was obtained in a purer condition than by crystallisation,
since fi'om a mixture flavone and aiithoc\-anin readily crystallise out
together.

Combustions of pure anthocyanin prepared from rose dore and from
bronze showed the anthocyanin pigments to be identical in both ca.ses,
the difference of colour in the flowers being merely due to the presence
of luteolin in the latter case.

In the same way combustions of pure magenta anthocyanin from
magenta and from crimson showed that crimson in the flower is a
mixture of magenta anthocyanin and luteolin. The same magenta
anthocyanin was also obtained from a variety — ivory tinged with
magenta — which is formed from magenta by loss of a deepening factor.
Hence the anthocyanin pigments in the tinged and full-coloured
varieties are identical.

From analyses of anthocyanin from both red and magenta varieties,
both pigments were found to contain much higher percentages of oxygen
than the flavone :

C

Red anthocyanin ... .'5r81°/„
Magenta anthocyanin .50-.50
Apigenin ... ... 66'66

A determination of the molecular weights of the two anthocyanins
showed them to be of the order of magnitude of 700 for magenta and
570 for the red. Hence, if derived from flavones, of which the molecular
weight is about 270, changes other than oxidation must be involved.
It is most probable that there is also condensation of two or more
molecules of flavone or condensation of a flavone molecule with other
aromatic substances. If the latter be the case, condensation with one

H

0 (by difference)

5-01 7,

4318 7„

511

44-39

3-70

29-64

M. Whbldalb

117

substance might produce red anthocyanin and a further condensation
with a second substance magenta anthocyanin, and the power to form
these substances would be represented by the red and blue factors.

Hjrpotheses in this connection are, however, of little value until we
have more knowledge of the constitution of the pigments themselves.
Nothing, moreover, can be said at present of the nature of the factors
causing the deepening or dilution of anthocyanin characteristic of deep
and pale varieties.

At this point some account must be given of the work of Will-
statter (36), on the anthocyanin of the Coi-nflower (Centaurea). The
author states that in the flower three pigments are present, a red,
a violet, and a blue. Willstatter is of the opinion that the anthocyanin
of Centaurea contains a nucleus of the type of Fig. 1, colour being due
to the presence of the quinonoid structure :

OH

Fig- 1.

The above would represent the nucleus of the violet pigment, which
is also in itself an acid. Under certain conditions a change readily takes
place to a colourless isomer, the oxonium oxygen becoming divalent :

Fig. 2.

The isomer is really a flavone with an extra and unusual hydroxyl
group in position 2.

The blue pigment in the flower, according to Willstatter, is the
potassium salt of the compound in Fig. 1, the position of the potassium
being uncertain. If the cell-sap is alkaline, the blue salt predominates
or is alone present.

118 Chemt'Mri/ of McnfleUan Factors for Flower-Colonr

If there is excess of acid in the cell-sap an oxonimn salt with an
organic plant acid is formed:

0-X

FiR. 3.

and crystalline salts, which the pigment readily forms with hydrochloi-ic
acid, are regarded as artificial compounds of a similar kin<l :

O-CI

HO

Fig. 4.

In the presence of excess of water, both the red and the blue forms
will also pass to the colourless isomer, but when a neutral salt, such as
sodium nitrate or chloride, is present the isomeric change is prevented
by the formation of additive compounds:

O-NO3

NaO

Fig. 5.

For preparing the pigment, the methods are long and elaborate, but
consist mainly in extracting the pigment from dried flowers by means
of water, sodium nitrate being added to jDrotect the pigment, and finally
precipitating the pigment with alcohol.

The blue pigment — cyanin — is a glucoside. On hydrolysis it is
decomposed into glucose and a pigment, cyanidine. Both cyanin and
cyanidine readily form crystalline compounds with hydrochloric acid.
Analyses of cyanidine chloride gave as simplest formula Q,^Ji.ySd.. HCI
but no account is given of any determination of the molecular weight.

The hypothesis advanced by Willstatter as regards the constitution
of anthocyanin is not at present based on any experimental evidence
beyond the existence of the salts with acids. The chief argument

M. WlIELDALB 119

against the hypothesis is that flavones, of tlie constitution indicated in
Fig. 2, are unknown.

No reference is made by Willstatter to the pigments in the actual
red and purple varieties of the Corn-tlower. If the pigments of the
varieties are identical with the red and purple pigments found in the
blue type, we must sujjpose the factorial differences merely to affect
the acidity and alkalinity of the sap, the sap of the red variety being
acid, of the purple, neutral, and of the blue type, alkaline, which does
not appear to be a very reasonable suggestion.

Grafe (13) also, working with flowers of scarlet Pelwrgonium, has
obtained an anthocyanin which forms very fine crystals, in combination
with acetic acid, of the composition, CisHosOis . 2CH3COOH. The
inheritance of colour in Pelargonium has not yet been investigated,
so that these results cannot be brought into use at present.

Factors involved in the Formation of the Anthocyanin pigments.

The next question to be considered is the nature of the factor, R,
connected with the formation of anthocyanin.

It was originally suggested (Wholdale, 30), that the R factor might
be an oxidase and that the formation of anthocyanin might be repre-
sented as :

glucoside + water ±^ flavone + sugar

X (flavone) + oxygen — *- anthocyanin

the first reaction being controlled by a glucoside-splitting enzyme, the
second by an oxidase.

Since the views in connection with oxidases and oxidase reactions
are very confusing, a short statement, as regards the action of oxidising
enzymes, may not be out of place at this point.

It has long been known that solutions of certain compounds, such
as guaiacum, a-naphthol, and benzidine are oxidised to coloured products
in the presence of plant extracts. The water extracts, for instance, of
some plants when added to guaiacum tincture at once produce a blue
colour (direct oxidase reaction). Extracts from other plants do not
produce this blue colour, unless hydrogen peroxide is also added
(indirect oxidase reaction). If a comprehensive examination be made
with these tests (Clark, 7, 8 ; Wheldale, 31), it will be found that the
members of many orders and genera {Umhelliferae, Labiatae, Boragina-
ceae and others) commonly give a direct action whereas the members

120 Chemistry of Mendel km Factors for Floiver-Cohmr

of other orders (Cruciferae, Caryophyllaceae, Crassulaceae, and others)
only as a rule give an indirect action. It will be noted at the same
time that the plants whose juices give the direct action, also turn
brown on injury, or when exposed to chloroform vapour. Also the
extracts themselves, on exposure to aii-, rapidly turn brown or reddish-
brown.

It is to Bach and Chodat(4) that, we owe the first suggestion that
;in oxidase consists of two elements, an oxygenase which oxidises some
substance to the state of a peroxide and a peroxidase which then
transfers the oxygen of the peroxide to oxidisable substances. The
hypothesis has been modified both by the authors themselves and by
other workers : the oxygenase has been eliminated and the more simple
hypothesis of a peroxide-peroxidase system has been retained.

It is now the opinion of various authors (Kastle and Loevenhart, 15 ;
Moore and Whitley, 20; Wheldale, 31) that those plants which give the
direct reaction form in their metabolism some substance which is able
to autoxidise and form an organic peroxide so that the system peroxide-
peroxidase is realisetl.

When, in the metabolism of the plant, no substance capable of rapid
autoxidation is formed, the system is not able to convey the oxygen of
the air to the artificial acceptor, i.e. guaiacum, but can only oxidise the
latter when hydrogen peroxide is added.

Hence the division of plant oxidising (nizymes into direct and
indirect oxidases is purely artificial. The factor of importance is the
peroxidase which is practically universally distributed and is the only
f;ictor which can be concerned in the formation of anthocyanin, for
anthocyanin is formed equally in jilants giving the direct or indirect
oxidase reaction, and moreover in a large number] of cases of plants
giving the direct action the albinos possess this power as well as the
pigmented types.

The view that anthocyanin may be formed from a chromogen by
the action of an oxidising enzyme has been supported by Keeble,
Armstrong and Jones (14, 16, 17, IS, 19). These authors, by means
of an excellent micro-chemical test for oxidases, have shown that there
is, in many cases, an intimate connection between distribution of
oxidases in tissues and the distribution of anthocyanin.

The above authors employed dilute solutions of a-naphthol and of
benzidine, in presence of hydrogen peroxide, as tests foi- oxidases, the
material used being chiefly colour varieties of fiowers and stems of
Primula sinenMS.

M. Wheldalb 121

The results obtained were as follows. Broadly speaking, in all
coloured varieties, jjeroxidases were found in the epidermis and bundle
sheathes, the tissues in which anthucyanin is distributed. Flowers of
the dominant white varieties were found to give oxidase reactions only
after certain inhibitors were artificially removed from the flower. Albino
varieties gave the oxidase reaction as fully as coloured varieties. Hence,
if the hypothesis that colour in Primula is due to the action of an
oxidase on a chromogen be true, we must assume that albinism is, in
this case, due to the absence of chromogen. Since the chemical nature
of the chromogen is unknown in Primula, this assumption can, for the
present, be made, though all Primula albinos certainly contain a flavone.

In the same way all albinos of the Sweet Pea and the Stock contain
both peroxidase and flavone, and here again, if the Keeble and Armstrong
hypothesis be con-ect, we must assume the absence of chromogen to be
the cause of albinism.

But in the case of Antirrhinum, peroxidase is present in both the
ivory parent and the white parent which on crossing produce magenta.
Since the evidence is strongly in favour of apigenin being the chromogen,
some third substance or reaction must be postulated and the high
molecular weights of the anthocyanins lend support to the hypothesis
that there is some further process involving condensation.

From the work of Keeble, Armstrong and Jones we can only at
present make the deduction that anthocyanin is formed in tissues
vi'here oxidation can take place, a;nd there is no evidence among the
well-known Mendelian cases of colour inheritance of a factor being
represented by a peroxidase.

Quite similar results to those of Keeble and Armstrong have been
obtained by Atkins (2, 3), working with flowers of many species and
varieties of Iris. The author shows that the distribution of peroxidases
and inhibitors is correlated with the natural colouring of the flowers.

The hypothesis that anthocyanin is formed by oxidation and con-
densation or by oxidation alone of the flavones is entirely refuted by
Combes (9, 10, 11, 12), who maintains that anthocyanin, on the contrary,
is formed by reduction fi'om flavones. His evidence may be summed
up as follows :

(1) There are two pigments in Ampelopsis hederacea, a yellow
(flavone) and a red (anthocyanin).

(2) The flavone can be converted into a red pigment, identical with
natural anthocyanin, by treating an alcoholic solution of the yellow

122 Chemistry of MendeUan Factoi'sfor Floiver-Coloiir

pigment with sodium amalgam in presence of hydrochloric acid. Hence
the anthocyanin is a reduction product of the flavone.

(3) The anthocyanin can be converted into a yellow pigment,
identical with the natural flavone, by means of hydrogen peroxide.
Hence the flavone is an oxidation product of the anthocyanin.

As regards the formation of anthocyanin from the flavone in
Ampelopsis, the method of procedure was as follows (Combes, 9) :

" La solution dans I'alcool a 90° du compos6 jaune brun, acidifiee
par I'acide chlorhydrique et soumise a Taction de I'hydrogene naissant
fourni par I'amalgame de sodium, prend j^rogressivement une coloration
rose violace qui devient de plus en plus foncde. Filtr^e et neutralisee,
la liqueur obtenue foumit par evaporation une substance pourpre.

Ce corps, comme I'anthoeyane naturelle extraite des feuilles rouges,
cristallise en aiguilles pourpres gi-oupees en rosettes. Apres deux
cristallisations dans I'alcool et trois cristallisations dans I'eau, le pigment
artificiel obtenu en partant du compose jaune natural, aussi bien que le
pigment anthocyanique naturel, commencent a se decomposer au bloc
Maquenne a 165° ; la fusion instantanee a lieu a 212° — 215°, tandis que
le compose jaune commence a se decomposer a 182°, et fond instantan^-
ment a 226°— 229°."

The artificial anthoeyaiiiu had, moreover, the same qualitative
reactions as the natural anthocyanin, i.e. it turned red on addition
of acid and green, becoming brown-yellow, with alkali. It gave a green
precifjitate with lead acetate and a green-black coloration with ferric
chloride. On addition of sodium bisulphite, the colour of the artificial
anthocyanin disappears, but reappears on acidification with sulphuric
acid, a reaction which is also characteristic of the natural pigment.

By the sodium amalgam method. Combes (11) was also able to
obtain, in several cases, artificial anthocyanin identical as far as qualita-
tive reactions are concerned with the natural product. The materials
used were :

(a) A crystalline yellow-brown j)igm('iit isolated from Privet
{Ligustrmn) leaves. The resulting anthocyanin crystallised in needles.

(h) A yellow-brown crystalline pigment isolated from leaves of a
variety of Vine which duos not redden naturally. Crystalline antho-
cyanin resulted.

(c) A crystalline pigment from Narcissus incomparabilis; the
resulting pigment had properties of anthocyanin.

M. Wheldale 123

(d) From artificially synthesised flavones (names not given). The
resulting pigment had the properties of anthocyanin.

As regards the conversion of anthocyanin into flavone by oxidation
the following method was adopted by Combes (10) : "Le pigment antho-
cyanique extrait des feuilles rouges Vigne-vierge (Ampelopsis hederacea),
purifie par deux cristallisations dans I'alcool et trois cristallisations dans
I'eau, et dissous dans I'alcool a 90°, donne una solution de couleur
pourpre, qui, additionnee de son volume d'eau oxygen^e, passe pen a
peu au rouge-brun, puis au jaune. De la solution jaune, ainsi obtenue,
j'ai extrait un pigment jaune-brun cristallis6 en aiguilles groupees en
rosettes."

The artificial yellow pigment was found to have the same projwrties
and melting point as the natural yellow pigment and was considered to
be a flavone.

In the absence of further evidence, it is difficult to avoid making
the following criticism of the experiment just quoted. Since no
mention is made of any methods employed in the preparation of
crystalline anthocyanin from Ampelopsis, it is highly probable that
this product contained flavone as impurity, since most careful purifica-
tion is necessary to remove all traces of flavone. Thus, from a mixture
of flavone and anthocyanin extracted from Antirrhinum, both pigments
will crystallise out together in plates which have, in spite of the presence
of flavone, a deep red colour. If the mixture from Ampelopsis were of
such a nature, on treating with hydrogen peroxide, the anthocyanin
would be decomposed though not necessarily into flavone, and the
flavone present as imjjurity could then be extracted from the resulting
solution.

A similar observation, however, to the above is made by Will-
statter(36), with regard to Cornflower anthocyanin. " Eine interes.sante
Umwandlung erleidet das Cyanidin, wenn wir seine alkoholische Losung
mit verdlinntem Wasserstiiffsupero.xyd anstatt mit Wasser erwarmen.
Nach der Entfarbung setzen wir einige Tropfen verdihinter Salzsaure
zu und erhitzen im Wasserbad weiter. Die Fliissigkeit wird gelb,
durch Extrahieren mit Ather lasst sich ein Produkt isolieren, das
schone, hellgelbe Krystalle bildet und mit Alkalien tief gelbe Losungen
liefert."

From the above one would make the deduction that, if the antho-
cyanin from Cornflower were pure from flavone, a flavone might be
formed by destruction, though not necessarily oxidation, of the antho-
cyanin.

124 Chemistry of Mendelicm Factors for Flower-Colour

This method was also tested on pure anthocyanin from Antirrhinum.
The pure pigment, prepai-cd by methods previously descrilied and
entirely free from flavone, was treated in a similar way with hydrogen
peroxide. The colour disappeared, giving a yellowish-brown solution
and precipitate, but from neither solution nor precipitate could any
flavone be extracted. From this it would appear that no flavone is
formed by the action of hydrogen peroxide on the anthocyanin of
Antirrhinum.

Observations of a similar kind to those made by Combes on the
effect of sodium amalgam on acid alcohol solutions of flavone.s have
also been made by Keeble, Armstrong and Jones (19). By treating
the alcoholic extracts of various flowers with zinc dust and hydrochloric
acid they obtained results which may be summarised as follows :

Ziuc and hydro-
chloric acid

AlkaU

Pale yellow (" primrose ") Wallflower

red solution

green

Yellow Daffodil

?

Yellow Crocus

?

Cream Polyanthus ...

?

Chinese Primrose

?

Primula sinensis (duiniiiant whitt,')

?

„ „ (recessive white)

extremely slight

?

Tsw(!tt (23, 24) also has obtained a red pigment by treating colour-
less extracts of apples with strong mineral acids in the presence of
formaldehyde or acetic aldehyde. A careful examination of this
artificial anthocyanin showed it to have very similar properties to
natural anthocyanin. The chief point of difference appears to be its
solubility in ether, for natural anthocyanin is insoluble in this medium,
unless, according to Tswett, it has stood with hydrochloric acid for
3 — 5 days. In addition Tswett obtained similar n'd pigments, though
he did not study them in detail, from pears (aldehyde not necessary),
white grapes, bananas, flesh of red grapes, white petals of roses and
Cyclamen. Negative results, however, were obtained with leaves of
white cabbage, mesophyll of red cabbage, Pelargonium leaves, orange
peel, petals of white pinks, white petals from buds of red pinks, flowers
of lily of the valley, carrots, potatoes, Kohlrabi, and barley seedlings.

The reaction which underlies some, if not all, of the above phenomena
is the following: when the alcoholic solution of some flavones is warmed
and acidified with strong hydrochloric acid and sodium amalgam or zinc

M. Wheldale 125

dust is added, an intense red or purplish-red coloration is produced
(Abderhalden, 1).

Hence the coloration given by many plant extracts in alcoholic
solution when treated with hydrochloric acid and sodium amalgam or
zinc dust is obviously due to the presence of Havone in the extracts, and
it is useless to postulate the nature of the reaction in the absence of
analyses of the red product.

The following points must then be considered:

(a) The question as to the identity of the artificial and natural
anthocyanins. The only characteristic reactions in common between
the two substances are the red colour with acids, the green colour with
alkalies and the decolorisation with sodium bisulphite, and it is possible
that these reactions might be given by two derivatives which were
similar, though not necessarily identical. The artificial anthocyanin,
after removal of alcohol and hydrochloric acid by evaporation, is soluble
in ether and in water, whereas natural anthocyanins are always insoluble
in ether and also almost insoluble in water excej)t when in the condition
of a glucoside.

(b) If the natural and artificial anthocyanins are not in any cases
identical, the reaction is still interesting as showing that coloured
products, very similar in properties to anthocyanin, can be obtained
by reduction from the flavones.

(c) If the natural and artificial anthocyanins are in all cases
identical, which is unlikely, then the reactions, by which the latter
are formed, must be very different frcjm those taking place in its
formation in the plant, since, for instance, ai'tificial anthocj'anin can
be produced, according to Combes, from members of the genus Narcissus,
which do not form natural anthocyanin. Artificial anthocyanin, on the
other hand, cannot be obtained from many species which do produce
natural anthocyanin.

(d) There is a possibility, however, that in some cases natural
anthocyanin may be formed by the treatment with nascent hydrogen,
as for instance in the case of plants which produce the anthocyanin
but in which the formation of pigment is inhibited in the flowers. It
is conceivable that the materials necessary for the formation of antho-
cyanin are present but there is inhibition for some unknown reason.
The treatment with sodium amalgam may remove the cause of inhibition
and natural anthocyanin may be formed. Only isolation and accurate

Journ. of Gen. iv 9

126 Chemistry of MendeUan Factors for Floiver-Colour

analyses of both artificial and natural antliocyauin from the same plant
can provide evidence which is of value.

CONCLU.SIONS.

1. There arc three varieties of Antirrhinum majus, ivory, yellow
and white which do not form anthocyanin. Ivory is dominant to yellow
and contains a factor "/" which is absent from yellow.

2. It has been shown that the pigments in the ivory and yellow
varieties are flavones. Ivory contains a pale yellow flavone, apigenin,
and yellow, in addition to apigenin, a deeper yellow Havone, luteolin.
Hence the "/ " factor may be represented as the power to inhibit the
formation of luteolin.

8. The white variety contains no flavone.

4. When either the yellow or ivory is crossed with a white of
suitable composition, an F^ containing anthocyanin is produced. There-
fore it appears likely that the anthocyanin is formed from a flavone by
the action of some factor contained in the white.

5. It has been suggested that anthocyanin is either an oxidation
or a condensation product of a flavone or both.

6. Two anthocyanins have been isolated from Antirrhinum, red
and magenta, the latter containing a "B" factor which is absent from
the red.

7. Anal3'ses of the red and magenta pigments have shown that
they both contain a higher percentage of oxygen than the flavones.
Also, magenta has a higher percentage of oxygen than red.

8. Determinations of the molecular weights of the red and magenta
pigments indicate that the anthocyanin molecules are at least twice or
three times as large as the flavones. Hence, in addition to oxidation,
condensation must have taken place, either between flavone molecules,
or between the flavone and .some other aromatic substance. In the
latter case, one substance, the " R " factor, may give red anthocyanin
and a second substance, the "B" factor, magenta.

9. The view that anthocyanin is in part, at anj^ rate, an oxidation
product is confirmed by the researches of Keeble, Armstrong and Jones,
who have shown that anthocyanin is formed in tissues most rich in
oxidising enzymes, though there is no good evidence, from the well-
known Mendelian cases, of albinism being due to loss of an oxidising
enzyme.

M. Wheldalb 127

10. From recent researches on the pigments of the Cornflower,
Willstatter states that the flower contains three pigments ; a purple
pigment which is an acid and owes its colour to the presence of a
quinone nucleus and which readily passes to a colourless isomer which
is a flavone derivative ; a blue pigment which is the potassium salt of
the purple ; and a red pigment which is an oxonium salt of the purple
with an organic acid.

11. As a result of recent work, Combes has brought forward the
hypothesis that anthocyanin is not an oxidation product but, on the
contrary, a reduction product of the flavones. As evidence, he quotes
experiments which bring about the formation of anthocyanin from
flavones by means of sodium amalgam in acid solution and the forma-
tion of flavones from anthocyanin by treatment of the latter with
hydrogen peroxide. The evidence, however, cannot be considered con-
clusive in the absence of analyses of the products.

12. A similar formation of artificial anthocyanin has been obtained
by Tswett, and by Keeble and Armstrong from plant extracts by treat-
ment with aldehyde and strong acid or nascent hydrogen in presence
of alcohol and strong acid. The artificial product has some of the
properties of natural anthocyanin but differs in its solubilities. Further
analyses are required for the determination of its identity.

BIBLIOGRAPHY.

1. Abderhalden, E. Biochemisches Handlexikon Berlin, 1911, Vol. vi.

2. Atkins, W. R. G. "Oxydases and their Inhibitors in Plant Tissues." Sci.

Proc. R. Soc, Dublin, 191.3, Vol. xiv. (N. S.), p. 144.

3. . " Oxydases and their Inhibitors in Plant Tissues." Part 2. " The

Flowers and Leaves of Iris." Sci. Proc. R. Soc, Dublin, 1914, Vol. xiv.
(N. S.), p. 157.

4. Bach, A., und Chodat, R. " Untersuchungen iiber die Rolle der Peroxyde in

der Chemie der lebenden Zelle." Ber. d. Chem. Ges., Berlin, various papers
in years 1902 — 4.

5. Badr, E. "Einige Ergebnisse der experimentellen Vererbungslehre." Bei-

kefte zur Med. Klinik, Berlin, 1908, Vol. x. p. 265.

6. . " Vererbungs- und Bastardierungsversuche mit Antirrhiimm." Zs.

indukt. Ahstamnnmgslehre, Berlin, 1910, Vol. ill. p. 34.

7. Clabk, E. D. The Plant O.vidases. Dissertation, New York, 1910.

9—3

1-28 Chemisti'ji of MendcUan Factors for Floioer-Colour

8. Clark, E. D. "The Nature and Function of tlic Plant Oxidases." Torreya,

New York, N.Y., 1911, Vol. xi. Nos. 2—5.

9. Combes, R. "Production exp(5rimentale d'unc antbocyanc identiquc a celle qui

se forme dans los feuilles rouges en automne, en jiartant d'un compose extrait
des feuilles vertes." C. R. Acad. Sci., Paris, 1913, Vol. CLVii. p. 1002.

10. . " Passage d'un pigment anthocyanique extrait des feuilles rouges

d'automne au pigment jaune contenu dans les feuilles vertes de la mtoie
plante." C. B. Acad. Sci., Paris, 1913, Vol. clvii. p. 1454.

11. . "Sur la presence, dans des feuilles et dans des fieurs ne formant pas

d'anthocyane, de pigments jaunes pouvant etre transforracs en anthocyane."
C. R. Acad. Sci., Paris, 1914, Vol. CLVIII. p. 272.

12. . " Untersuchuiigen iiber den chemischen Prozess der Bildung der Antho-

kyanpigmente." Ber. d. hot. Ges., Berlin, 1913, Vol. xxxi. p. 570.

13. Gbafe, V. " Studien iilier das Anthokyan." 3 Mittheilung. Sitzber. Ak.

Wiss., Wien, 1911, Vol. cxx. p. 765.

14. Jones, W. N. "The Formation of Anthocyan Pigments of Plants." Part 5.

"The Chromogens of White Flowers." Proc. R. Sue, London, 1913, Vol.
Lxxxvi B, p. 318.

15. Kastle, J. H. and Loevenhakt, A. S. " On the Nature of Certain Oxidi-sing

Ferments." Amer. Ghem. Journ., 1901, Vol. xxvi. p. 539.

16. Keeble, F. and Armstrong, E. F. " Tlie Di.stribution of Oxyda.scs in Plants

and their Role in the Formation of Pigments." Proc. R. Soc, London, 1912,

Vol. Lxxxv B, p. 214.
17. . "The Bole of O.xydases in the Formation of the Anthocyan

Pigments of Plants." Jimrnal of Genetics, 1912, Vol. ii. p. 277.
18. and Jones, W. N. "The Formation of the Anthocyan Pigments

of Pl.iiits." Part 4. "The Chromogens." Proc. R. Soc, London, 191.3,

Vol. i.xxxvi B, p. 308.
1!). . "The Formation of the Anthocyan Pigments of Plants."

Part 6. Proc. R. Soc, London, 1913, Vol. Lxxxvii B, p. 11.3.

20. Moore, B. and Whitley, PI " The Properties and Classificiition of the

Oxidising Enzymes, etc." Uiochem. Journ., 1909, Vol. iv. p. 1.36.

21. Perkin, a. (r. Various papers in Ch.em. Soc. Trans, from 1895 — 1904.

22. Smiles, S. The Rehitions hetveen Chemical Constit%Uion and some Physical

Properties. London, 1910.

23. Tswett, M. " Heitriige zur Kenntnis der Anthocyane. Ueber kiiii.stliches

Anthocyan." Biochemische Zeitschrift, Berlin, 1913, Vol. lviu. p. 225.

24. . " Zm' Kenntnis des 'vegetabilischen Chamaoleons.'" Ber. d. hot. Ges.,

Berlin, 1914, Vol. xxxii. p. 61.

25. Wheldale, M. "The Inheritance of Flower Colour in Antirrhinum majus."

Proc. R. Soc, London, 1907, Vol. lxxix B, p. 288.

26. . " Further Observations on the Inheritance of Flower Colour in Antirrhi-
num maJus." Reports to the Evolntion Committee of the Royal Society, London,
1909, Report V. p. 1.

M. Wheldale 129

27. Wheldale, M. " The Colours and Pigments of Flowers with special Reference

to Genetics." Proc. R. Soc, London, 1909, Vol. Lxxxi B, p. 44.

28. . "On the Nature of Anthocyanin." Proc. Phil. Soc, Cambridge, 1909,

Vol. XV. p. 1.37.

29. . " Die Vererbung dar BlUtenfarbe bei Aniirrldnum majiis." Zs. induht.

Ahstammungslehre, Berlin, 1910, Vol. in. p. 321.

30. . "On the Formation "f Anthocyanin." Journal of Genetics, Cambridge,

1911, Vol. I. p. 1.3.3.

31. . "On the Direct Guaiacum Reaction given by Plant Extracts." Proc. R.

Soc, London, 1911, Vol. LXXXlvB, p. 121.

32. . " The Flower Pigments of Antirrhimim majus. L Method of Prepara-
tion." Biochem. Journ., Cambridge, 1913, Vol. vii. p. 87.

33. and Bas.sett, H. Ll. "The Flower Pigments of Antirrhinum majus.

II. The Pale Yellow or Ivory Pigment." Biochem. Journ., 191.3, Vol. vir.
p. 441.

34. . "The Chemical Literpretation of some Mendelian Factors tor

Flower Colour. Proc. R. Soc, London, 1914, Vol. Lxxxvii B, p. 300.

35. . "The Flower Pigments of Antirrhinum majus. III. The Red

and Magenta Pigments." Biochem. Journ., Cambridge, 1914, Vol. viii. p. 204.

36. WiLLSTATTER, R. and Everest, A. E. "Ueber den FarbstofFder Kornblume."
Liebigs Ann. Chem., Leipzig, 1913, Vol. 401, p. 189.

A COMMENTARY ON THE GENETICS OF
THE CILIATE PROTOZOA.

By CLIFFORD DOBELL.

PREFACE.

For many years the ciliate Protozoa have been favourite objects of
study, and consequently they have furnished niucli curious information
concerning many problems of genetics. This information is, however,
to a large extent still unincorporated into current biological conceptions.
It has therefore seemed to me that it would not be an altogether
thankless task to attempt to extract from the immense and often hardly
accessible literature dealing with this group of organisms, such facts as
are likely to interest workers in the field of genetics ; and to present
these facts in a summary fashion easily comprehensible to the reader
unversed in protozoology. This is my aim in the following pages.

From its very nature, therefore, it will be apparent that I do not
aim at setting forth in this article anything more than a selected body
of facts extracted from the works cited in the appended bibliography —
a consideration which I would ask the writers of these works to bear in
mind, if they should find much that they have contributed to our know-
ledge passed over in silence, or treated in cavalier fashion. Without
rigid and critical selection it would have been impossible to compress
all the accumulated facts of many years into anything less than several
bulky volumes.

In the presentation of the facts I have adopted the following plan.
I have begun with a very brief general account of the Ciliata — in order
to make clear some essential peculiarities of these organisms : I have
then given a short account of the classical researches of Maupas' — since
current conceptions concerning the Ciliata are largely based (explicitly

' I may remind the reader that the work on the Ciliata before the time of Maupas has
bpen exhaustively dealt with by Biitschli (1887-89).

132 On the Genetics of the Ciliate Protozoa

or implicitly) on these : I have then reviewed and commented upon the
work which has been done since his day : and finally I have drawn a few
of the more obvious conclusions — conclusions which will appear to many,
I am afraid, the least satisfactory feature of this article. Siiife the works
of many of the older investigators — Blitschli, Balbiaiii, I'^iigelmann, and
the rest — are in many ways no less admirable and important than the
more modern researches of R. Hertwig, Enriques, Jennings, Popofif,
Woodruff, and others, I have more than once referred to these; but
such references have been relegated to footnotes.

To facilitate the numerous cross-references in the body (if the papei-
I have adoj)ted the plan of numbering the paragi-aphs. A parenthetic
reference thus (§ x) signifies that on refeniug to the paragraph
numbered x, the reader will find additional information relating to
the matter under discussion.

I am fully conscious of having made many omissions, but I trust
that uiisstatements concerning the facts are few. Such criticisms as
I have ventured to make are based upon my personal knowledge of the
ciliates, and I should be the first to welcome corrections or counter-
criticisms.

A general analysis of the subject-matter of this article will be found
on page 183. I have added it to facilitate reference to the subjects dealt
with : and I have placed it at the end because I should prefer the article
to be read as a whole before special sections of it are consulted. My
commentary aims at being a consistent whole, and its several parts —
considered singly — may therefore appear obscure or incomprehensible.
If the reader's curiosity has been sufficiently excited, he will accordingly
proceed now to the first chapter.

CHAPTER I.

Tlie Organization of a Ciliate, and the Chief Events in its Life.

1. A typical ciliate possesses a peculiar organization, which is often
misunderstood. Its body is built upon the plan shown in Fig. 1 (A).
It is clothed, more or less, with locomotory ciliary appendages (c) often
greatly differentiated. The animal possesses a mouth {mo.) defining
the ventral surface ; and as one end is specialized as a " head " end, we
can further distinguish anterior and posterior, dorsal, and right and left
lateral regions of the body — which is generally asynnnetric. In the
protoplasm there are usually vacuoles (/('.) in which food is digested.

C DOBELL

133

and also one or more rhythmically contractile vesicles (c.v.) serving
probably for excretion or for regulation of the water-content of the
protoplasm. Many (jther organs may be present.

2. The characteristic nuclear apparatus consists of two nuclei or
systems of nuclei — a meganucleus (M), and a micronucleus (m). The
latter may be represented by one, two, or many nuclei ; whilst the
ixieganucleus is frequently segmented, or otherwise modified.

3. Reproduction is usually effected by transverse fission of the body
into two. The meganucleus is divided by a simple constriction, but the
micronucleus (or micronuclei, as the case may be) divides mitotically.
(See Fig. 1, B.) Unequal bipartition or budding occurs in some forms.

anil

".no.

m. ._

/.:

A

4. Most ciliates are known to be able to encyst, and so withstand
the drying of the liquid in which they live, or other adverse conditions.
In some forms temporary cysts are formed during fission.

5. Most ciliates perform from time to time a peculiar act called
conjugation. The details of this process vary considerably in different
forms, but the following is an approximately true general account of
the events occurring during a typical conjugation'. (See Fig. 2, A — H.)
The organisms unite in pairs, the protoplasm subsequently fusing and
becoming continuous at the point of contact (Fig. 2, A, B). Each

1 This account is based on the cases of Colpiditim colpoda (Maupas, 1889) and Para-
mecium caudatvm (Calkins and Cull, 1907), which are perhaps as "typical" as any other
cases, and are fairly simple because there is only a single micronucleus and meganucleus
in these forms.

134 On the Genetics of the Ciliate Protozoa

individual's micronucleus (or one of them in a multimicronucleate
form) now undergoes two' successive mitotic divisions, forming thus
four micronuclei (Fig. 2, B). Three of these four nuclei degenerate
(Fig. 2, B, C) and the remaining one again divides into two — a
"stationary nucleus" (s.n.) and a "migratory nucleus" (in.v.). Each

mn I

rn 71 2

D

n

Fig. 2.

migratory nucleus now passes into the opposite individual of the pair,
and fuses with its stationary nvieleus (Fig. 2, C, D, E). Each individual
thus comes to have a single micronucleus (z.n.) which is the product
of its own stationary nucleus and the migratory nucleus of its partner
(Fig. 2, E). The two organisms now separate, and their meganuclei
degenerate and disappear (Fig. 2, E). At the same time, the micro-
nucleus (fusion nucleus, s.n.) divides thrice in succession — producing
eight daughter nuclei (Fig. 2, E, F) in each individual. Of these eight
nuclei four increase in size, becoming new meganuclei, four remain
small as new micronuclei (Fig. 2, F, G). By two successive transverse
fissions of the whole organism, four small individuals are subsequently
formed (Fig. 2, G, H), each with the nuclear arrangement characteristic

' In souie forms tliree or more such divisions occur.

C DOBELL 135

of ordinary individuals'. They grow and divide subsequently in the
ordinary fashion (§ 3).

6. In Vorticella and its allies, conjugation of a different type
occurs^ The two conjugating individuals are of different size — a large
and a small, respectively sessile and motile. As in the previous case,
they come in contact and fuse — their meganuclei sooner or later
degenerating. The micronucleus in the larger individual divides twice
successively by mitosis, giving rise to four nuclei, of which three
degenerate and disappear. In the smaller individual the micronucleus
undergoes three successive divisions, thus producing eight nuclei, of
which seven degenerate. The two conjugating individuals now fuse
completelyl Their two remaining nuclei divide once more, one of the
two daughter nuclei so formed in each degenerating, the other persisting.
The latter then come together and fuse. The single uninucleate in-
dividual resulting from conjugation then reorganizes its nuclear system
in a manner comparable with that described above (§ 5) — the details of
the process probably being different in different forms'".

7. A " reduction " or halving of the chromosomes (meiosis) has been
shown to occur during the micronuclear divisions which take place at
the beginning of conjugation ^ The halving appears to occur usually

' The details of the process whereby the new nuclear apparatus is constructed after
conjugation may be very different from those outlined above. In all cases, however, the
reorganization is of essentially the same nature — the formation of a new meganucleus and
micronucleus from the single fusion nucleus.

- This account is based upon V. vionilata (Maupas, 1889).

3 According to Maupas (1889) the membrane clothing the body of the smaller in-
dividual is cast off and degenerates — it is not absorbed into the body of the larger.

* The accounts given above are conformable with the views of nearly all those workers
who have studied the conjugation of the ciliates since the time of Maupas. It should be
stated, however, that this accepted version of the sequence of nuclear events has been
questioned by some workers, Hoyer (1899) believed that in Colpidium no nuclear fusion
occurs during conjugation — the migratory nuclei are simply exchanged, and the stationary
nuclei then degenerate. The new nuclear apparatus is therefore formed, in each in-
dividual, entirely from the nucleus which it receives from its partner. More recently,
Dehorne (1911, 1911a) has stated that the same thing happens in Puramecium, and that
he has confirmed Hoyer's account of Colpidium. Dangeard (1911, 1911a) has contradicted
this in the case of the latter form (wrongly called Colpoda in his first paper), and main-
tains that the accepted version is correct. I believe that Hoyer and Dehorne are wrong in
their contention. Maupas, R. Hertwig, Calkins and Cull, and all other recent workers
who have studied conjugation, are unanimously agreed that a nuclear fusion does occur.

' Chromosome reduction has not been shown to occur in Paramecium. Calkins and
Cull (1907) believe that the chromosomes are halved at the first micronuclear division,
but they are "far too numerous to count." They judge them to be approximately 165 in
number at this stage, whilst at other divisions the number is "greater than this." In
Stentor (2 species) the chromosomes appear to be reduced to a quarter — not a half — of the
usual number during the "reduction" divisions (Mulsow, 1913).

186 On the Genetics of the Ciliate Protozoa

at the second division. (See Praiidtl (1906), Enriques (1907), Pop.ift"
(1908 a), etc.)

8. It is possible that a ciMlain ainninil nf (_-vti)])lasiii is always
exchanged during conjugation'. This has hccii dcsi-iihcil in at least
one form — Stentor. (See Mulsovv, 1913.) In the parasitic t'orni Aiio-
•plophrya, according to Collin (1909), each conjugating individual receives
half the meganucleus of its partner-. After the exchange, however, it
degenerates and di.sappears. The meganucleus is not known to behave
in this manner in other forms.

9. After this brief account of divisicm and conjugation", we may
consider the interpretation to be put upon these events and the com-
parisons which may be drawn between them and the conditions which
obtain in other animals. Failure to make a correct comparison, confusion
of ideas, and misuse of words, have unfortunately introduced a great
many unnece.s.sary complications into this relatively simple matter.
A real analysis of the vital phenomena of the Ciliata is possible onl}'
when we discard the erroneous a priori deductions of the modern cell
doctrine and certain evolutionary speculations — with all their mis-
conceptions and slipshod phraseology — and consider these organisms
objectively. I have pointed out elsewhere how this may be done, so
that it will be unnecessary to repeat what I have already wi'itten.
(See Dobell, 1911.) I shall now merely state a few obvious homologies
between a ciliate and a metazoon which are fundamentally important
for a comprehension of what follows. These homologies are as correct
as I believe it possible to make them with the words at my command.
But I would emphasize here that the very peculiar organization and
behaviour of the ciliates — which are in many ways radically different
from all other organisms — render all descriptions of them, in terms
borrowed from those other organisms of a different order, only approxi-
mately true.

10. First, a ciliate is not unicellular — it is not the homologue of
one cell in the body of a metazoon, but is homologous with an entire

' For tliose who regard the chondriosomes, and not the chromosomes, as the "bearers
of hereditary characters," it may he of interest to add that chondriosomes have been
described in many different ciliates, bnt their behaviour during conjugation has not yet
been studied.

- First described in Anoplophryu by Schneider, in 1886.

'■' It should be stated that the conjugation here described is that characteristic of most
free-living ciliates. In some parasitic forms quite different processes occur. I may
mention Oixdina, which forms small ciliated gametes (Neresheimer, 1907; Metcalf, 1909),
and IcIUIiyuphthirius, which exhibits a peculiar form of self-fertilization (Neresheimer,
1908; Buschkiel, 1911).

C. DOBBLL 137

metazoon. The ciliate is a non-cellular but complete organism : it has
a body formed on the same fundamental plan as that of a metazoon,
but it possesses no cellular differentiation. Cytoplasm and nuclei are
specialized for special functions, but they are not sub-divided into
separate units or " cells."

11. Secondly, it is clear that the moganuclear system of a ciliate
is the equivalent of the nuclei of the somatic cells of a metazoon ; and
the micronuclear system of a ciliate is the equivalent of the germ-cell
nuclei of a metazoon. These homologies — first made clear by Maupas
(1889) — rest upon the observed behaviour of the nuclei during conjuga-
tion, and at other times in the life-history. It seems to me unnecessary
to adduce here all the evidence which can be used to establish this
proposition.

12. Thirdly, conjugation is a process of reciprocal fertilization (in
the cytological sense) ; but it is coupled with a process of complete
reorganization' unlike anything known to occur in other organisms.
The conjugating individuals may be suitably called conjugants, and
those which have just conjugated and separated, exconjugants. It is
obvious that the conjugant is not a gamete, but a sexual individual:
and the exconjugant is a zygote — but of a very remarkable kind ; for it
is the remains of the " parent " organism reorganized after the addition
of a foreign nucleus.

13. Fourthly, the migratory nucleus is a microgamete nucleus and
the stationary nucleus a macrogamete nucleus. This is evinced by their
behaviour before and during conjugation : for the micronuclear divisions
which precede conjugation (§ 5) are homologous with the maturation
divisions of metazoan germ-cells (§ 7), and the active migratory nucleus
is in some (?all) cases of smaller size than the stationary nucleus.
{Didinium (Prandtl, 1906), Paramecium (Calkins and Cull, 1907), etc.)

14. Fifthly, organisms which — like Faramecium — conjugate in the
manner described in § 5, must be regarded as hermaphrodite" individuals
at the time of conjugation : for they each produce the equivalents of
a " male " gamete and a " female " gamete. Organisms which conjugate
like Vorticella (§ 6), however, are of a different sexual nature. The
large conjugant is a female individual, producing the equivalent of a
" female " gamete. The small conjugant is a male individual, producing
the equivalent of a "male'' gamete. There is an additional comjalication

' First definitely stated by Engeimaim (1875).

'' A conclusiou drawu by Engelmanu (1875) and earlier wurliers, but from incorrect
premisses.

138 On the Genetics of the Cilia te Protozoa

here, moreover, beeause the ciinjiigants as well as their gamete nuclei
fuse during conjugation, so that only one complex zygote results. It
seems to me necessary to lay special emphasis on the preceding state-
ments, because the terms " gamete," " sex," " male " and " female " are
almost always misused in the case of the ciliates, and a very great deal
of confusion has arisen in consequence'. It is manifest that " male
sex " or " female sex " is properly predicated of individuals, which are
ultimately known by the gametes which they produce. To suppose
that a " male gamete " is itself a male is absurd. This misleading term
really signifies " a gamete produced by a male individual." The gamete
itself has no sex — within the legitimate meaning of the word.

15. Lastly, it should be clearly realized that among the ciliates the
sole method of reproduction is by division. Sexual "reproduction" (in
the sense of " multiplication ") does not occur". The zygote is a con-
jugant reorganized — a new individual raised upon the ruins of an old,
without any increase in the number of individuals. After conjugation,
the exconjugant reproduces by fission (the earliest divisions being
sometimes modified, cf. § 5) in essentially the same manner as any
other individual. All " genei'ations " are produced asexually — by fission
or budding. The only " sexually produced generation " is the excon-
jugant, which is merely the conjugant reorganized — an individual
thrown into the melting pot, with a bit of another individual, and
recast. In the case of the Vorticellids (§ 6), two whole individuals,
as well as their gametes, fuse to form one zygote ; so that sexual
" reproduction " in these forms results in a reduction — not increase —
in the number of individuals.

To convince himself of this, the reader need only refer to almost any of the recent
works dealing with genetic experiments on ciliates. He will find that the same author
assumes a conjugant to be a cell, a gamete, or a sexual organism, at pleasure — according
to the requirements of the argument. Examples of such inconsistencies could be given
by the dozen.

- "Ce n'est pas, en effet, un des resultats les moins surprenants des recherches sur
la f^condation des Cilifa, de voir que son Evolution et ses manifestations si complexes
n'aboutissent a la multiplication on a la reproduction d'aucuu etre nouveau " (Maupas,
1889, p. 435).

C. DOBELL 139

CHAPTER II.

The Life-cycle of a Ciliate according to Maupas.

16. I propose in this chapter to state very briefly the chief general
conclusion.? drawn by Maupas from the extensive investigations which
culminated in his great works of 1888 and 1889. His clear and precise
conceptions will form a convenient fixed point from which to contem-
plate the work which has since been done.

17. Maupas claimed to have proved that the lives of ciliates run
in cycles'. There is first a period of asexual multiplication (agamic
period, or period of immaturity), during which the progeny of an
exconjugant continue to multiply, without conj ugating . or being able
to conjugate. At the end of this period — of definite length for each
species — the organisms attain the age of puberty, after which con-
jugation is possible and normally occurs in nature. Unless they
conjugate, the organisms continue to multiply for a further definite
number of divisions. This second period — during the whole of which
the animals are sexually mature — is called by Maupas the eugamic
period. If, for any reason, conjugation does not occur during this
period, the animals now enter a third period of multiplication — the
period of senescence — which is also of definite duration. Throughout
this period the animals suffer more and more froin old age, until they
finally die after completing a given number of fissions. Those organisms
which are able to conjugate successfully during the eugamic period
become " karyogamically rejuvenated," or reorganized, and start another
cycle of development like that just described.

' This view was first definitely expressed — so far as I am aware — by Claparede and
Lachmann (18(50) and by Balbiaui (1860). It has been stated by Woodruii (190!1 a) that
Dujardin "maintained that the life-history of infusoria comprises a cyclical change iu
vitality which terminates in death." I know of no passage in his writings which can
be construed in this fashion. He certainly did not believe in a "cycle" such as that
described by Maupas, for he held that a sexual act had not been proved to occur in any
ciliate. Dujardin (1841, p. 87), suggested that there is possibly a limit to multiplication
by fission, but he did not maintain this proposition or take sides on the issue. Balbiani's
statements, however, are very definite. He says that he has established the fact that
there is a limit to asexual reproduction in the ciliates. The period of asexual multiplica-
tion terminates with natural death of all individuals belonging to the same cycle ; or " by
a return of sexual reproduction, indicating the completion of one of these cycles and the
beginning of a new cycle"; or by encystation. (See Balbiani, 1860.) His views were
therefore at this time — he frequently changed them — very similar to those of Maupas.

140 On the Genefirs of the Ciliate Protozoa

18. A concrete case may be given in illustration. The life-cycle
of Styloiujchia pustulata may be tabvilated thus' :

Divisions.
1 — Excon j ugant.

: [ Period of immaturity (agamic period).
130 — Age of puberty.
: • Eugamic period.
170

: [ Period of senescence.
316— Death.
The length of one complete cycle for this species is thus 316 divisions,
but Maupas found that the cycle is of diiferent length in different
species. For example, Sti/luni/cliia mi/tili(s died of old age after 319
successive bipartitions, O.rytriclni and Urii/chodromus (jrandis after
320 — 330, Leucophrys patula after 660.

19. The period of time occupied by a developmental cycle depends,
of course, upon the rate at which division takes place. This, according
to Maupas, depends upon four factors : (1) the individual temperament
of the species ; (2) the biological adaptation of the species to its form
of nutrition; (3) the quality and quantity of the nutrition^ ; (4) the
temperature. Light and other external factors he found to have no
appreciable effects.

20. Three conditions are, according to Maupas, necessary for
successful conjugation. These are: (1) hunger; (2) sexual maturity;
(3) diversity of ancestry of the conjugants ("f^condation crois^e") — that
is, the conjugants must nut be related-'. "External physical conditions
play no determining part in the appearance of conjugation ^" Hunger —

' The division at which the eugamic period ends and the period of senescence begins
is variously stated by Maupas to be the ICOtli and tlie 170th — 180th.

- Earher experiments had been made by Balbiani (1860) in this connexion. He states
that be placed three individuals of Paramecium aurelia separately in three different watch-
glasses. In one of these he placed (i drops of pure water, in another 6 drops of a pepper
infusion containing many bacteria and amoebae, in the third 3 c.c. of the same infusion.
After 16 days, the first animal had divided once, and both the daughter individuals died.
After a similar length of time he found the second animal had jiroduced 17 offspring :
whilst the third had produced in 17 days so numerous a progeny that he estimated them
at approximately 2100.

■■I If they are, their union is sterile, or produces weakly progeny which ultimately die.

■* Biitschli (1887) after analysing all the evidence avadable at the time, had concluded
that "external conditions which call forth conjugation have not yet been determined."

C. DOBBLL 141

caused by transferring animals from a culture containing plenty of food
into one containing none or little — induces conjugation, provided that
the animals are " karyogamically mature," and unrelated. In cultures
containing closely related animals it brings about encystation or abortive
attempts to conjugate. If the organisms are too richly nourished during
the eugamic period, conjugation does not occur.

21. Senescence, which occurs in the last period of the life-cycle in
organisms which have failed to conjugate, is manifested by the following
phenomena : decrease in the size of the organisms, degeneration of the
nuclear apparatus (in various ways), reduction of the mouth-parts and
appendages, physiological degeneration (loss or inhibition of various
functions), and sometimes by " morbid sexual hyperaesthesia," leading
to abnormal and sterile conjugations between closely related individuals.
The final result is always death.

22. Maupas did not share the view — which had then recently been
revived by Weismann' — that the Ciliata are "immortal." He believed
that his researches had shown that these animals are not capable of
multiplying by fission for an endless period. Unless conjugation takes
place at the proper season, the doom of the organism or its offspring
is sealed, and old age and death are as inevitable for Parcmiecimn as
for man.

CHAPTER III.

The Results of Later Investigators.

23. Having briefly outlined the conclusions reached by Maupas,
I shall now review the chief results which have accrued from the
investigations of subsequent workers. For the sake of convenience,
I shall sub-divide these results into two main groups — according as
they concern (A) the asexual or (B) the sexual period in the life-history:
and I shall consider the experimental results, in each subject studied,
as far as possible in chronological order.

' Weismann's conception of the "immortality" of the Protozoa was familiar to earlier
investigators — e.g., Ehrenberg and Diijardin. The first expression of this idea with which
I am acquainted is in the work of the former (Ehrenberg, 1838). Moreover, he clearly
realized the allegorical nature of this "immortality" — "welche poetisch genug an Unsterb-
lichkeit und ewige Jugend grenzt. " "Man theile sich in zahllose neue Theile, um
zahllose Jahi-e zu leben und jung zu seyn." Woodruff (1909 a), however, is wrong in
stating that Ehrenberg held that the "Protozoa are so simply organized that they are
not subject to natural death." Calkins (1902a) is also wrong in stating tliat "Ehrenberg's
view was vigorously contested" by Dujardin. (Cf. also footnote to § 17, supra.)

Journ. of Gen. iv 10

142 On the Genetics of the Ciliate Protozoa

A. The Asexual Period.

24. We may first consider the duration of the period of multiplica-
tion by fis.sion. It is important to inquire how far Maupas was justified
in concluding that this period is of a limited duration, and that — ■
without conjugation — old age and death form the natural termination
of the ciliate life-cycle.

25. The first experiments to be considered are those of Joukowsky
(1898), who kept four cultures — each started from an exconjugant — of
Pleurotricha lanceolata. One of these continued for 458 generations',
without conjugation or degeneration. This line was still developing
normally when it was abandoned. Another line persisted for 250
generations, a third for 442 — both finally dying. The fourth was
followed to the 220th generation. It developed further, but abnormally.
Two similar cultures of Paramecium, caudatum reached 150 and 170
generations, and then died. Conjugation was observed twice, and
degenerate forms were found at the end of the time. The first culture
{Pleurotricha) is interesting, on account of its long duration ; but from
the others no definite conclusions can be drawn.

26. Kulagin (1899) studied Paramecium, and suggested that
" senescence " in old cultures is really due to the accumulation of
harmful substances in the bodies of the organisms or the surrounding
medium. Satisfactory evidence for this conclusion he does not give.

27. Simpson (1901) found that in his cultures of Paramecium the
rate of division of the organisms gradually decreased, until they finally
died. He did not attempt to ascertain the determining factors.

28. Calkins (1902 (with Lieb, 1902), 1902 a, 1904), also investigated
Paramecium, and made a similar observation. He succeeded in keeping
one line for 742 generations, without conjugation occurring; others for
shorter periods. In all cases he found that the rate of fission gradually
became slower, and the animals entered a '-period of depression." From
this they were able to recover if stimulated with various chemicals and
extractives. " The well-marked cycles, with periods of depression which
demanded stimulation of a decided character, were approximately of six
months duration, while intermediate cycles of le.ss importance were
about three months long" (Calkins, 1904). "One natural way in

' It should be remembered that all "generations" are asexually produced among the
ciliates. The term does not connote any of those secondary meanings associated with
the conception of "generation" in sexually reproducing organisms.

C. DOBELL 143

which the weakened descendant may be restored to new vigor is
by conjugation with another individual" (Calkins and Lieb, 1902).
"A couple of hundred generations, more or less, uses up the potential
of vitality, whereupon, unless the potential is renewed [by conjugation
or stimulation], the race dies out with some indications of protoplasmic
old age" (Calkins and Lieb, 1902).

29. Calkins (1904) denied that the meganucleus increases in size
during depression periods, or that the micronucleus degenerates (contra
Maupas). " Temperature changes have little or nothing to do with the
decline of vigor" (Calkins, 1902). Depression is not morphological, but
physiological, and is shown by pathological divisions and the formation
of monsters, decrease in the rate of division, and death. The recovery
from depression — brought about by chemical stimuli — he regarded as
a case of " artificial parthenogenesis." This is based, apparently, upon
the erroneous supposition (§§ 12, 14) that the conjugant — or would-be
conjugant — is a gamete.

30. R. Hertwig has formulated a hypothesis to account for the
"depression" periods observed by himself, Calkins, and many others,
in cultures of ciliates. It is involved in his " theory of the karyoplasmic
ratio " (Kernplasmatheorie) which may be stated as follows : the mass-
relation of nucleus to cytoplasm expressed as the quotient ^ — that is,

the mass of nuclear substance divided by the mass of cytoplasm — is
a ratio whose magnitude is of fundamental importance for all vital
processes influenced by the nucleus, for assimilation and organizing
activity, for growth and division (R. Hertwig, 1908, p. 5, et alibi —
cf. 1899, 1902, 1903, 1903 a, 1905).

31. Hertwig (1899, 1903, 1903 a), from studies of Paramecium,
Dileptus, and other Protozoa, believes that depression is due to the
development of an abnormal karyoplasmic ratio. During a number
of successive generations, the nucleus gradually increases in size at the

expense of the cytoplasm — in other words, ^ becomes larger. The

resulting disproportion causes depression, or inhibition of vital activities.
It may be compensated by elimination or absorption of nuclear substance,
or by conjugation — both of which are considered to restore the karyo-
plasmic ratio to normal : or it may be so great a disproportion that the
animal is unable to recover, and consequently dies. Hertwig found
that the meganucleus actually is disproportionately large in animals
in a state of depression.

10—2

144 On the Genetics of the Ciliate Protozoa

32. Popoff (1907) has obtained results which he interprets in a.
similar manner. He cultivated a lino of Stylonychia mytilus for
3^ months. During this time he observed "depression periods" — like
those of Calkins (§ 28) — which became deeper and deeper until the
culture finally died out. In depressed individuals he found the mega-
nucleus abnormally large (§§ 29, 31 ), and often fragmented or of irregular
shape : the micronuclci frequently multiplied, and the whole organism
was smaller (§ 21). No conjugations were observed', though depre.ssed
animals were "inclined to conjugate." They recovered from depression
by resorption of the excess of chromatin in the meganucleus (?) — thus
restoring the karyoplasmic ratio to normal (§ 31). Similar dejiression
periods were caused in Paramecium by overfeeding. Recovery occurred
as a result of conjugation. The morphological changes observed were
similar to those seen in Stylonychia; and PopotT sees "a complete
parallel " in the nuclear behaviour during depression with that seen
during conjugation. " Everything goes to show that the cause of
depression is not to be sought in accidental changes in environment
(such as food, water, etc.), but that it lies in the organism itself."

33. In a series of papers, Enriques (1903-1910) has maintained
that the " depression " or " senescence " seen in cultures of ciliates is
not due to "old age" or any internal cause, but is merely the result of
adverse environmental conditions — the most important being overgrowth
of bacteria. " Depression " is due to prolonged poisoning with bacterial
toxins. If these are not allowed to accumulate, and the culture medium
is frequently renewed, no depression occurs, and the animals are capable
of multiplying asexually ad infinitum. The chief evidence advanced by
Enriques in support of the last proposition is the statement that he has
kept Glaucoma, in cultures for 683 generations, without it conjugating
or showing an}- signs of " depre.ssion " or " .senile decay " (1905). Other
organisms kept for shorter periods gave similar results — e.g. Oxytricha,
Stylonychia, Vorticella (1903, 1905 a, 1910, etc.). "Agamic reproduction
can be continued as long as one likes, if the technique is good and
bacteria are not too numerous. No change of food is necessary" (1909rt).
Enriques strongly criticizes the work of Calkins (§ 28) and Popoff
(§ 32).

34. In his later papers Popotf (1909 a, 1909 i) has shown that
"depression" such as he observed in his cultures (§ 32) may be brought
about by treating the animals with certain chemicals. He cultivated

' Possibly because the animals were all descended from the same ancestor? (§ 20).

C. Do BELL 145

Stylonychia in water containing CO.j and Paramecium in water
containing ammonia. In both he observed increase in the size of
the meganucleus, followed by its fragmentation, multiplication of the
micronucleus, and loss of power to divide or assimilate food. The
nuclear changes are compared with those occurring during conjugation,
but the animals treated in this manner never conjugated. Similar
effects were not produced by other chemicals (NaCl, MgCL, MgSOj,
glucose). Popoff interprets these results as indicating that katabolic
products accumulate during a depression period and cause a disorganized
condition of the organism which can be corrected by conjugation and
its concomitant reorganization (§ 31).

35. Results similar to those of Popoff (§ 34) have been obtained
with Paramecium by Sun (1912), by adding uric acid or calcium
phosphates to the culture liquids.

36. Gregory (1909) has kept a line of Tillina, magna for 548
generations'. "Depression periods" were observed, but chemical
stimulation had little effect — " rejuvenation took place, but only to
a slight degree." The culture finally died, without conjugation
occurring. Gregory interprets her results in the same terms as
Calkins (§ 28). She denies that the relative size of the meganucleus
increases during depression : but the conclusion appears to rest upon
the use of a fallacious " coefficient," and it appears from the few measure-
ments given that her results do not really contradict Popoff's (| 32).
Moody (1912) has obtained closely similar results with Spathiditnn,
which she cultivated for 218 generations. Death, however, was prob-
ably due to " abnormal conditions of the environment." She also
denies that the size of the nucleus increases during depression, but
her measurements do not bear this out. She employs the same
peculiar " coefficient " as Gregory-. Calkins, it may be added, has
succeeded in keeping Blepharism.a for 224 generations, and Actinoholus
for 446 generations. Both lines then died. (See Calkins and Gregory,
1913.)

37. The most extensive work on the duration of the asexual period
has been done by Woodruff. Woodruff's earliest work (1905) was on
the hypotrichous forms Oxytricha, Pleurotricha, and Gastrostyla — the

' This organism divides into 2 or 4 daughter-individuals inside a temporary cyst. In
one place (p. 399) the number of generations is given as 546.

- The statistics given for Actinoholus (Table 5) are to me absolutely unintelligible.
The volume of the cytoplasm is given in every case as less than that of the nucleus (which
is inconceivable) ; yet the average ratio of nucleus to cytoplasm is found to be 1 : 20.

146 On the Genetics of the Ciliate Protozoa

longest lines of which he succeeded in keeping for 860, 44'8 and 288
generations respectively. He observed Calkins's "cycles" in division
rate (§ 28), with corresponding " depression periods " resulting finally
in death. "Rejuvenescence" was brought about by beef extract
(Oxijtrichn). "The number of generations which constitute a cycle
is not at all constant," and the "cycles" themselves show secondary
cycles or "rhythms" — i.e. minor fluctuations in division rate. No
conjugations occurred.

38. Quite recently Woodruff (1913 6) has published some measure-
ments of individuals from his long line of Oxytricha. He finds that the
mean size of both the organism and the nucleus is " smallest at periods
of high reproductive activity, and becoines progressively larger as the
division rate falls." During depression periods there is an increase in
the relative amount of cytoplasm as compared with nucleus (§§ 31, 32).
As Woodruff appears to have selected his individuals at random, at
unknown inter-division moments, it is not surprising that he finds the
karyoplasmic ratio very variable'. This consideration seems to me to
render his measurements of very questionable significance.

39. In a long series of papers, Woodruff (1908 a-1914)'' has reported
upon his investigations of Paramecimn aurelia'. "This race of Para-
mecium has attained so far (May 1, 1912) 3029 generations during the
five years it has been under daily observation. The number of genera-
tions attained during each of the first five years of its existence is as
follows : first year 452, second year 690, third year 613, foiirth year 612,
and fifth year 662. The mean rate of division for the entire period is
over three divisions in forty-eight hours. Periods of marked physio-
logical depression have not occurred — such variations in the rate of
reproduction as have appeared being either normal rhythms or the
effects of environmental changes of temperature and culture medium.
The organisms of the present generation are in as normal morphological
and physiological condition as the original ' wild ' individual isolated
to initiate the culture" (Woodruff, 1912). Later (Woodruff, 1913) we
learn that 3340 generations have been reached : still later (Woodruff,
1913a) "more than 3800 generations" in "over 6 years"; and finally
it is announced (Woodruff, 1914) that the 4102nd generation has been

' See § 47 infra.

'■' Summaries of the work of Woodruff and his collaborators will be found in Woodruff'
(1912 a) and Middleton (1913).

' P. caxidatum has given comparable results on a smaller scale. Concerning the
specific distinctions of these two farms see Woodruff (1911 b). Compare also § 78 infra^

C. DOBELL 147

attained'. Conjugation has never occurred in this line'-. It is con-
cluded that the " cycle " of Calkins (§ 28) " is probably an artificial one
which is brought about by the subjection of the race to an environment
which is not suitable for its prolonged existence " (Woodruff, 1911 d).

40. Woodruff' and Baitsell (1911, 1911 ct) have found that "when
P. aurel'ia is bred on a varied culture medium, or on a constant culture
medium of beef extract, cycles do not occur, but rhythms (§ 37) persist.
It is not possible by constant environmental conditions to eliminate the
rhythms." The "constancy" of the environment in these experiments
is, however, not above suspicion.

41. Woodruff (1911, 1911 a) has proved that the excretion products
of Paramecium have " a decidedly depressing effect on the rate of repro-
duction," and this no doubt accounts to some extent for depression
periods in cultures. " These excretion products are essentially specific
in their action" (1913a). This result should be compared with those
of Enriques (§ 33), Popoff' (§ 34) and Sun (§ 35). The last named
investigator has found — like Woodruff — that culture fluid in which
Paramecia have been growing is toxic to other Paramecia : but she
has made the curious observation that the toxicity is lost if the fluid
be boiled.

42. Baitsell (1912) has succeeded in cultivating several lines of
Stylonychia pustulata for a large number of generations. The longest
line ended at the 572nd generation (cf Maupas, § 18). " Conjugation
never occurred and abnormal or degenerating animals did not appear,
but after a gradual decline in the fission rate the culture finally died
out." This result was pi-obably not due to " the ending of any definite
life-cycle," but to unsuitable cultural conditions. More recently,
Baitsell (1914) has come to the same conclusion from studies of
Occytricha and Pleurotricha.

43. If we consider the results obtained by all the investigators
mentioned in the preceding paragraphs, it seems highly probable that
"depression" or "senescence" inciliates is due to unhealthy surroundings
— unsuitable food, toxins produced by the organisms themselves or their
cultural companions, etc. — and is not due to any inherent inability to
live or frustrated necessity to conjugate. Woodruff's conclusion con-
cerning Paramecimn seems equally applicable to all other ciliates : " It
is probable that most, if not all, normal individuals have, under suitable

' Dr Woodiuff informs me that the number of generations now reached (March 20,
1914) is 4310.

'' But see § 101 infra.

148 On the Genetics of the Clliate Protozoa

environmental conditions, unlimited power of reproduction without con-
jugation or artificial stiiindation " (Woodruff, 1911 d).

44. Concerning the morphological manifestations of " depression "
there is not complete unanimity among investigators. For whereas
some believe that depressed organisms are of smaller size (Maupas,
Calkins, Popoff), others maintain that they are larger (Woodruff,
Gregory) : and similarly some observers find an increase in the relative
size of the raeganucleus (Hertwig, Popoff, Sun), others a decrease
(Calkins, Moody, Woodruff). Some of these conclusions cannot be
accepted unreservedly. It seems on the whole probable that hyper-
trophy of the meganucleus often accompanies depression : and I have
little doubt that the discordant conclusions result in pjirt from the
loose application of the terms "depression" or "senescence" to a variety
of different conditions.

4-5. We may now consider some remarkable work by Popoff (1908)
on what may be called the mechanics of division. The aim of his
investigations was to test the truth of one of R. Hertwig's deductions
from his " Kernplasmatheorie " (§ 30). According to Hertwig, the
normal karyoplasmic ratio of an organism (or cell) becomes gradually
altered owing to the growth of nucleus and cytoplasm at unequal rates,
until a condition of " karyoplasmic tension " is finally set up. This
tension, or unbalanced condition, is the factor which determines division
of the organism (or cell) as a whole — the division giving rise to new
individuals in which the karyoplasmic ratio is once more normal.
Division is thus regarded as a regulatory reaction to an abnormal
mass-relation between nucleus and cytoplasm.

46. Popoff's earliest results (1908)' were obtained with Frontonia,
a large holotrichous ciliate of symmetrical and regular shape suitable
for measuring. It possesses a single meganucleus — upon which the
measurements were made — and several micronuclei. Cultures were
kept at various temperatures, and the volume of nucleus and cytoplasm
computed from measurements made on animals at known intervals of
time between one division and the ne.xt. The following results may be
selected as illustrating Popoff's general conclusions. (See Fig. 3.)

47. At a temperature of 25' C. Fmntoiiia divides once every
17 hours, during which time the cytoplasm grows at a uniform rate
(Fig. 3). The nucleus, however, begins immediately after division to
decrease slightly in volume — probably owing to loss of liquid during

' These experiments were beguu origiually by Wierzbizki, another of Hertwig's pupils
(Hertwig, 1905).

0. DOBELL

149

its recovery from the preceding division. At the second hour, however,
the nucleus begins to grow, but it does so more slowly than the cyto-
plasm. Accordingly, the karyoplasmic ratio (pj gradually increases.

P

In the just divided organism, the "karyoplasmic norm" y^ = 67; but

after 15 hours' growth this has increased to 100. The period from the
2nd to the 15th hour is called by Popoff the "period of functional
growth of the nucleus," and at the end of it the karyoplasmic ratio
reaches its maximum — the " moment of karyoplasmic tension." During

Frontonia Uucas. ZS'C.

Time (hou.rs). ^^

Fig. 3. (From Popoff, 1908, modified.)

17

the next two hours, the nucleus grows ("division growth") more rapidly
than the cytoplasm, so that at the I7th hour the karyoplasmic ratio is

once more normal ( -^ = 67 j. Division now takes place — each daughter-

, „ ™ , ,, , 1 • X- P volume of cytoplasm ,-,■ „ . • ,

' Popoff reckons the karyoplasmic ratio as ^,= ^ ; , which is Hertwig s

A volume of nucleus

karyoplasmic ratio inverted (§ 30).

150 On the Genetics of the Giliate Protozoa

individual possessing at its formation a normal karyoplasmic ratio. And
so the cycle of events continues.

48. Pojjoff' (1909) obtained confirmatory results with I'arainecium.

P

The value of .-> and the rate of division vary with the temperature in

both cases; but the general conclusions given above appear valid for all
experiments, mutatis mutandis.

49. The " moment of karyoplasmic tension " is the moment at
which division is determined. If this were not so, indeed, it would
be difficult to understand how any division could occur'. Popotf ( 1 908)
established this by ingenious vivisection experiments. The karyo-
plasmic ratio of a Frontonia was changed by cutting ofif a piece of its
cytoplasm. If this was done during the period oi'fitiietiunal growth, the
cytoplasm continued to grow until the normal karyoplasmic ratio was again
established. Division was consequently delayt^d, but finally took place
in the usual way. If, however, a piece of cytoplasm was removed during
the period of division growth, the animal did not regulate its karyo-
plasmic ratio by compensatory growth of cytoplasm ; but it divided at
the proper time (I7th hour) with an abnormal karyoplasmic ratio. This
seems to indicate that the moment of karyoplasmic tension determines
division. And, moreover, Popoff elicited the curious fact that the plane
of division is also determined at this moment : for an animal from which
cytoplasm had been removed during the period of division growth
divided, into two unequal-sized individuals, at the plane which would
have been median had the animal been whole.

50. Moody (1912) has made .some measurements of Spathidium
which — she believes — tally with Popoff's results for Frontonia and
Paramecium (§§ 47, 48). But unfortunately no real confirmation of
his laborious and important researches has yet appeared. It is easy
to criticize or disbelieve his conclusions, but to test them by further
experiments performed in the same manner is an arduous task. And
without further experimental evidence, criticism of this work becomes
merely an academic discussion of probabilities — of no real value.

51. Inquiries have been instituted into the effects of chemical and
physical agents upon the rate of fission in ciliates, and some results of
this work may suitably be considered here. We have already seen

' In the case cited above, for example, it will be seen that the karyoplasmic ratio is
normal (67) just before division. As it is the same in the uewly divided organism, one
naturally asks why division should occur at all — assuming it to be determined by the
karyoplasmic ratio. Some such explanation as that given above therefore becomes
necessary.

C. DOBELL 151

(§§ 28, 36, 37) that certain chemical (and physical ?) stimuli are able
to increase the rate of division in " depressed " cultiires\ but similar
effects have also been pi-oduced in normal organisms.

52. Peters (1904) has laid emphasis on the fact that the salt
content of the medium in which ciliates are cultivated is a most
important factor. He found as a result of numerous experiments that
an excess of KCl in an otherwise normal medium accelerates the division
of Stentor. Small quantities of chloroform act similarly upon Para-
mecium.

53. Calkins and Lieb (1902) who studied the effects of alcohol on
"depressed" Paramecium found that this substance acts, in a certain
concentration, as " a continued stimulus which sustains the high rate
of division even during periods of depression of the control series." But
Woodruff (1908) has shown that minute doses of alcohol sometimes
increase and sometimes decrease the rate of fission. In the former case,
" the effect is not continuous, but gradually diminishes and finally the
rate of division falls below that of the control." A larger dose of alcohol
will again increase the rate of fission, but again for a limited period
only-. These results may be compared with those of Daniel (1909)
who finds that Stentor and Spirostomum can acquire a specific resistance
to alcohol — becoming " acclimatized " to small but gradually increasing
doses. Both Woodruff and Daniel found that alcohol-resistant ciliates
are more susceptible to certain other chemicals.

54. The toxicity of certain salts to Paramecium has been studied
by Woodruff' and Bunzel (1909). Estabrook (1910) has studied the
effects of various chemicals on the growth of the same form. All the
substances tried^ behaved in a similar manner: " When very weak they
have no effect whatever. In greater concentrations, all retard the later
stages of growth" and cause "other injury to the organism." It is
surprising to find, however, that complete growth can occur in a medium
consisting of nothing but distilled water and a little NaCl. It may be
noted here that Piitter (1905) has found that Paramecium and other
ciliates can live for many days in distilled water containing no free
oxygen.

55. Many of the earlier workers found that temperature has a
marked influence on the rate of fission (§ 19). All later workers have

' And the decrease in rate of fission during "depression" may also be due to chemical
causes.

- Cf. also Matheny (1910).

^ These were NaCl, nicotine, strychnine, and alcohol, in different concentrations.

152 On fhe Genetics of the Ciliate Protozoa

reached the same conclusion. [Cf. Joukowsky (1898), Popoff (1908),
Rautmann (1909), Woodruff and Baitsell (191 U), Sun (1912), etc.]
The higher the temperature, the more rapid the rate of fission. For
example, Popoff (1908) found that Stylonychia niytilus divides about
once every 8 hours at 25° C, every 16 hours at 18° C, every 30 hours
at 14° C, every 48 hours at 10° C. Woodruff and Baitsell (19116),
Sun (1912) and Jollos (1913) point out that the increase in the rate
of division with increase of temperature follows van't Hoff's rule for the
velocity of a chemical reaction.

56. There is, of course, a maximal and a minimal temperature
beyond which a ciliate cannot live and divide. These limits were
determined by Rautmann (1909) for Puramecimn as 35° C' and 5°C.
respectively. More recently Hutchison (1913), who has studied several
forms, concludes that each species " has a resistance jDeculiarly its own,"
but " the amount of variation within the species may be considerable."
Different strains of Paramecium caudatwm, foi' instance, showed very
different powers of thermal resistance.

57. Rate of fission may also depend to some extent upon the form
of nutrition (§ 19). An illustration of this is given by Joukowsky
(1898) who found that Pleurotricha reproduced more rapidly when
fed carnivorously on Uroiiema thari when kept in infusions of hay,
flour, or albumin.

58. It was found by McClendon (1909) that the rate of fission in
Paramecium is accelerated if the animal is subjected to centrifugal
force for a certain time. His controls, however, seem to have been
abnormally slow in dividing, and the mortality excessively high.

59. According to Peters (1904) KCl not only accelerates division
in Stentor (§ 52) but also modifies its character. The organism under-
goes an abnormal process of " budding " often forming very minute
" dwarfs." Further information about this curious phenomenon is to
be desired. The tlwarfs seem to have originated by what may be
called "chemical vivisection " or mutilation of the individual. It would
be interesting to know whether they are viable and able to reproduce —
whether they reorganize to normal size and form — whether they produce
dwarf or normal offspring.

60. According to Joukowsky (1898) the size of a ciliate may depend
upon its food. Pleitrotricha was found to vary from 200 /j. to ]5 /x in

' It was found that a temperature as high as 45^ C. might be withstood if the animals
were exposed to it for only a very short time. Compare also Jollos (1913).

C. DOBELL 153

length — giant individuals of this species and Oiiychodronius resulting
from cannibalism.

61. We may now pass to a consideration of variations and their
hereditary transmission — a matter to which the foregoing facts inevit-
ably lead us. The nature of " heredity " in the asexual reproductive
process of a ciliate should be clearly realized, and I would therefore
remind the reader of a few important facts before introducing this
subject. It is often assumed that " inheritance " in a ciliate is a very
simple matter — one organism merely dividing into two exactly similar
organisms. A moment's thought, however, will make it clear that when
a complex creature with morphologically and physiologically differ-
entiated ends divides transversely across the middle, the acquisition
of the parent's form by the two daughter-individuals is by no means
so simple a matter as at first sight it might ajjpear. Indeed, it is
probably true that all the complex buccal and appendicular structures
— in fact the entire ciliary coat and all its derivatives — of the parent
are resorbed or undifferentiated during division and a set of corre-
sponding organs formed by new growth and differentiation in each
daughter organism. Details of this process in certain complex forms
have been worked out especially by Wallengren (1901) and Griffin
(1910). In no known case is the differentiated protoplasm of the
parent passively parcelled out or " handed down " to the offspring in
the fashion contemplated by a priori speculators. Only certain
inclusions' (food, symbiotic algae, etc.), behave thus. The nuclei, of
course, divide during division of the organism as a whole, but in the
meganucleus undiff'erentiation and subsequent re-differentiation often
occur during fission.

62. Simpson (1902) has shown that when a Paramecium divides
into two, the products — measured at given later moments — may differ
from one another in size. But Jennings (1909) has shown that "these
'variations ' are mere temporary fluctuations, without effect in heredity "
(§ 63).

63. Jennings (1908 a, 1909, 1911) has now demonstrated, by long
series of careful measurements and observations, the important fact
that the species of Paramecium called caudatum and aurelia are not
homogeneous — that each consists of an assemblage of distinct races
(Jordan's " little species," Johannsen's " pure lines ") which differ inter
se, but which in se are constant. These races differ from one another
not only in size and form but also in physiological respects. The

' But see § 81 et seq., infra.

154

On the Genetics of the Ciliate Protozoa

distinctive characters of 6 such " pure lines " of cnudatum. and 5 of
aurelia are fully set forth by Jennings and Hargitt (1910).

64. The nature of these pure lines will be instantly envisaged in
the accompanying diagram (Fig. 4). We here see (upper line) outlines

X340

of individuals of mean size belonging to eight different races of Para-
mecium, drawn to the same scale. The differences in size are remarkable
— the actual mean length of the largest race (L., caudatum) reaching
230 /ti, of the smallest (i, aurelia) only 90^.

65. Within each race the individuals may differ considerably from
one another in size — as a result of growth, nutrition, and external
conditions. This is shown diagrammatically in Fig. 4 (lower line),
which depicts the relative dimensions of 10 individuals belonging to

C. DOBBLL 155

the race D (upper line). The largest individual has a length of 256 /x,
the smallest 80 ix. But the races all breed true to their mean dimensions.
" Breeding from the extreme specimens — ;the largest and smallest — of
a .single race, we get several hundred individuals from each. Both
produce progent/ of the same mean size. Each pi'oduces a whole series
of varying individuals, just like the original racial series ; the series
produced by the largest individual is exactly like that produced by
the smallest, or by any other. Selection within the pure race is of no
effect on the size" (Jennings, 1909). All the races appear to be
" singularly resistant, remaining quite constant in most respects, so
far as has been determined" (Jennings, 1911). The importance of
these conclusions for every worker engaged in the study of genetics
in the Ciliata is obvious. There can be little doubt that Jennings's
conclusions will be found to hold good for ciliates other than Para-
mecium. He himself (cf. Jennings and Hai-gitt, 1910) has shown that
there are many indications pointing to this conclusion : and he has also
made it clear that non-recognition of the existence of pure lines in
general populations of ciliates is a source of error which can lead — and
probably has led — to many wrong conclusions concerning variation in
these organisms.

66. Many interesting observations have been made upon the pro-
duction and asexual transmission of variations, and the more important
of these may now be considered. We may begin with certain normal
"modifications" which are due to differences of temperature. R. Hertwig
found that the value of the karyoplasmic ratio (§ 30) in a given species
of ciliate varies according to the temperature at which the organisms
are cultivated'. As a general rule, the size of the meganucleus is
relatively larger in animals kept at a low temperature, smaller in those
at a high temperature. Further, this difference is both relative and
absolute, for the organisms themselves are larger at a low temperature
than at a high temperature.

67. Confirmation of Hertwig's conclusions (§66) has been given by
Popotf (1908, 1909) and Rautmann (1909), for Frontonia, Stylonychia,
and Paramecium. An illustration may be taken from the case of
Stylonychia (Popoff, 1908), summarized in the following table ^:

■ The value of the karyoplasmic ratio also appears to be different in different races of
Paramecium. See note by Popoff and Rautmann (Popoff, 1909, p. 180).

^ L, B and W stand for length, breadth and width. The length of the nucleus is the
combined length of its two components, the width the mean of the two widths. The units
of measurement are not microns but di%'isions of the ocular micrometer used by Popoff.
I have given the measurements to the first decimal place only.

156 0)1 the Genetics of the Ciliate Protozoa

Mean

Dimensions of

Body

L.

Meganucleus

iperature C".

L.

B.

W.

B.

\V.

25

9-7

6-6

4-5

4-6

0-9

0-8

17—19

11-4

8-7

5-3

5-7

10

11

10

12 ■«

9-0

6-3

5-8

1-3

1-2

The karyoplasmic ratios f -^j calculated for these three temporatiires

are respectively 807, 77'4, 74. The dimensions of the micronuclei in
Fruntonia appear to vary like those of the meganucleus (Popofif, 1909),
but in Stylonychia they were not studied.

68. These size-differences are not permanent ; apparently they are
manifested as a direct resjjonse to temperature — like differences in rate
of fission (§ 55). Rautmann (1909) has found, however, that ri.se of
temperature, rise of fission-rate, and rise of karyoplasmic ratio do not
all run parallel. For increase in rate of fission (in Paramecium)
accompanies increase in temperature — the temperatures tried being

5° to 35° C. : whereas the karyoplasmic ratio f-v^j increases with the

temperature up to 25°, but then begins to sink. When an organism
is subjected to a change of temperature, it can regulate its karyoplasmic
ratio to that characteristic for the new temperature in the space of
time which elapses between one division and the next — for a tem-
perature interval of 5° C.

69. Jollos (1913) experimenting with pure lines of Paramecium,
cuudatum, has found that although differences in size are produced by-
changes of temperature — as described by Hertwig, Popofif and Rautmann
— nevertheless these differences are transitory. At a new temperature,
a change of size is at first observable, but later it disappears. The
animals appear to become adapted to the new temperature and then
to readjust themselves to their original proportions. Jollos also states
that resistance to extremes of temperature can be induced in Para-
mecium to a slight degree. But here again the modification is
impermanent — being lost if the organisms are returned to a normal
temperature.

70. In one case Jollos (1913) claims to have produced a permanent
change (" mutation ") in Paramecium. From a race subjected to a high
temperature he obtained a small race which is permaveutly small — at
high, normal, and lower temperatures. This race has conjugated without
reverting to the original size. It differs also in being less susceptible

C. DOBELL 157

than the original race to sudden changes of temjDerature. The original
culture was derived from a single individual; and after conjugation an
exconjugant was similarly isolated and cultivated, so that Jollos's belief
that he has assisted at the production of a " mutation " appears' to be
justified.

71. Jollos (1913) has further recorded experiments with arsenic
compounds. He finds that Paramecium may acquire an increased
resistance to arsenic if treated with it for some time-. An arsenic-
resistant race so produced remains resistant for a considerable time if
bred further in an arsenic-free medium. But the increased resistance
gradually diminishes in the course of time, until it is finally lost. The
loss is more rapid if the animals are subjected to sudden changes in
temperature and nutrition. For such a partially permanent change
Jollos proposes the name of " enduring modification " (Dauermodifika-
tion). (See also infra, § 113.)

72. Estabrook (1910) has found no evidence that a race of Para-
mecium of a given mean size can be transformed by chemicals (§ 54)
into a larger or a smaller race. Temporary changes in size were
observed, but regarded as due to " variations in the nutritive and
other conditions of the normal environment." Concerning another
ciliate, however, Prowazek (1909) has published a peculiar observation.
Leucophr-ys patula was said by Maupas to be dimorphic — some organisms
being large, others small ^ Prowazek has confirmed this : and he adds
that he has been able, by treating cultures of the smaller form with
minute doses of quinine, to extract from them races of the larger form.
These, however subsequently reverted to the smaller form.

73. Among the records of experimentally produced variations the
researches of Popoff occupy a prominent place. From studies relating
to the karyoplasmic ratio (|§ 45 — 49) he surmised that variations in
size might originate through alteration of the relative proportions of
nuclear and cytojDlasrnic matter. Thus if an organism with a given
karyoplasmic ratio ;*■ chanced to divide into two new individuals of
unequal size, but each with a normal ratio («) ; then it might be
supposed that the now individuals would give rise to new races of

' His paper is only a preliminary note, without adequate evidence for his statements.

2 Compare the similar but more definite results previously obtained by others with
trypanosomes — reviewed in an earlier paper in this Journal (Vol. ii. p. 201, 1912).

3 Maupas thought the small forms were probably sexual individuals. Prowazek never
saw them conjugate, and appears not to share this view : but what his own interpretation
of the dimorphism may be (? sexual) I cannot comprehend. It is certain, at all events,
that the large and small individuals are not females and males (§ 14).

Journ. of Gen. iv H

lofi On the Genetics of the Ciliate Protozoa

larger or smaller individuals. Popoff believes that ho has actually
observed this in Stentor and Frontonia (1909) and Stylonijchia (1908).
As a result of unequal division, large and small races arose. They both
had a normal karyoplasmic ratio, but differed in size. (See Fig. 5,
which shows the relative sizes of a large (A) and small (B) race of
Htentor — the animals being outlined at the moment of division. A large
(C) and small (D) race of Frontonia is similarly compared — the animals
outlined immediately after division.) Unfortunately, it is not clear fi-om
Popoff"s statements that he actually saw the unequal divisions which

Fig. .5. (From Popoff, 1909.)

are supposed to have originated these new races. And the criticism
of Jennings' that Popoff" may merely have isolated pure lines (§ 63)
from a mixed population is one which seems both plausible and cogent.
74. More convincing are Popoff"s other experiments (1909) on
Stentor. This ciliate possesses a meganucleus of a form comparable
with a string of beads. By centrifuging an animal which was about
to divide, Popoff' caused the nucleus to be unequally distributed to the
two daughter-individuals — one receiving 16 "beads," the other only 3,
whilst the latter animal was similarly only a quarter of the size of the

1 See Jennings and Hargitt (1910).

C. DOBELL 159

former. Both individuals reorganized themselves successfully after
fission, and continued to multiply normally for about a week'. They
produced giant and dwarf races respectively, according to expectation.
It was also ascertained that the individuals of the smaller race had
reorganized their meganuclei so that they consisted of the normal
number of " beads."

75. In another experiment Popoff (1909) suddenly cooled a Stentor
which had begun to divide. The division was thus made to regress.
Placed at a norma! temperature, the animal then reorganized itself in
a peculiar way into a single individual. It then gi-ew to a very large
size, and subsequently divided. A race of giant Stentors was obtained
in this way which continued to divide and breed true for as long as the
culture was kept (about li months). [The size of this race can be
gauged from Fig. 5, E, which shows a dividing individual drawn to the
same scale as the other Stentors, A and B.] The karyoplasmic ratio of
the giant individuals ajjpeared to be the same as that of the normal
race from which they were derived. One of the giant Stentors from
the race just described is said to have divided unequally into a large
and a small individual. From the former, an even larger race was bred.
All attempts to obtain still larger races failed.

76. Popoff (1909) tried to produce new races of Stentor by cutting
off pieces of protoplasm from an individual and so changing the karyo-
plasmic ratio. The experiments were inconclusive, though it is stated
that a small piece of a Stentor containing a small piece of nucleus can
reorganize itself into a small individual capable of dividing several
times — always forming small individuals. Death always followed, how-
ever, and no small race was produced in this fashion'-. It is to be
hoped that all Popoff's experiments will soon be repeated by other
workers.

77. Somewhat similar experiments have been made on Paramecium
by Calkins (1911) and Peebles (1912), and the latter concludes: "The
removal of a portion of the cytoplasm does not result in the production
of smaller individuals. After several generations have been produced
the normal size is regained."

1 The cultures were then lost.

- Kegeneration has, of course, been studied in some detail in Stentor — e.g. by Balbiani
(1888, 1892, 1893), Gruber, etc. But I know of no record of the production of a race of
different size as a result of mutilation. It seems probable, moreover, that vivisected
Stentors sooner or later reorganize their meganuclei so that they consist of the normal
number of "beads.'' (Cf. Balbiani (1888, 1893), Prowazek (1904), etc.)

11—2

160 On the Genetics of the Ciliate Protozoa

78. The two species of Paramecium called avreiia and caudatum
differ fioin one another, inter alia, in that the former has two micro-
nuclei, the latter one. [See Maupas (1.S88, 1889), Hertwig (1889).]
Calkins (1906), however, advanced the view that both forms belong to
the .same species. Of a pair of exconjiigants of caudatum. (1 micro-
nucleus) he found that one reorganized after conjugation as a caudatum
form, the other as an aurelia — with 2 micronuclei. The aurelia form
persisted for a number of generations, but finally reverted t(j the
caudatum type. Jennings and Hargitt (1910) and Woodruff (19116)
nevertheless regard aurelia and caudatum as " good " species. In this
I agree, and I think the conclusions of the former explain Calkins's
results — " in rare cases specimens of the caudatum. races have two
micronuclei, those of aurelia, races but one'."

79. It may be noted here that Sun (1912) has observed divisions
in Paramecium in which the nuclei arc abnormally distributed — the
daughter-nuclei all remaining in one individual whilst the other receives
none. Enucleate and supernucleate forms may thus arise. The foi-ma-
tion of similar enucleate Stentojs has been observed by Prowazek (1904).
Individual Paramecia containing only a micronucleus have been found
by Kasanzeff (1901) and Sun (1912). The former found them in starved
cultures, and believed that they arose by an irregular division or
through " hunger degeneration " of the meganucleus. No evidence
of the production of new races in this fashion has yet been brought
forward.

80. Abnormally nucleate races oi' Paramecium have, however, been
experimentally produced by Lewin (1910). By cutting an individual
transversely through the meganucleus, he obtained two organisms, one
of which retained the micronucleus whilst the other had none. The
latter continued to divide, thus producing an " amicronucleate " race.
In another case, Lewin cut an abnormally dividing organism so that
one-half contained a meganucleus only, the other a meganucleus and
both daughter-micronuclei. The former gave rise to an "amicronucleate"
race — multiplying slowly, but normally : the latter produced a race with
two micronuclei. Both bred true for a considerable time-.

' It is perhaps worth noting here that Powers and Mitchell (lOlll) have described what
appears to be another species of Paramecium (called P. multimicroniu-leutuin) which re-
sembles candatum but possesses from 2 to 7 micronuclei.

■^ Le Dautec (1897) had previously stated that ciliates deprived of their micronuclei by
cuttinK in this manner are able to regenerate this organ. A full account of his experiments
has never been published, nor has any confirmation of his statements yet appeared.

C. DOBBLL 161

81. Teratological variations' are not very uncommon in ciliates,
especially in old cultures. One naturally wants to know the genetic
behaviour of monstrous characters, and fortunately some information
has already been elicited. Balbiani (1893, p. 56, PI. II, figs. 44, A— N)
was, I think, the first to describe the inheritance of an abnormality.
He cut a Paramecium, aurelia transversely, and the posterior individual
of the two so formed regenerated and later divided. " In the course of
the fourth generation an abnormal prolongation in the form of a horn
developed on the anterior individual." It persisted during seven
subsequent generations, passing to the anterior daughter-individual
at each fission, but gradually moving further backwards. Finally it
passed to the posterior end of its last possessor, which then died. The
sister-organisms appear to have been normal and " hornless " in every
case.

82. A comparable case was recorded by Simpson (1901). Of four
descendants of a normal exconjugant Paramecium caudatwiu three were
normal, but the fourth " developed a cleft tail." This animal continued
to divide for several generations. The abnormality persisted in the
posterior individual at each division, becoming gradually modified from
a "cleft tail" into a long "dorsal lobe." After eight divisions, the
abnomial animal died. In every case the anterior sister-individuals
were normal.

83. More extensive investigations of the inheritance of such
abnormalities have been undertaken by Jennings (1908). He isolated
abnormal Paramecia" (with "horns" or "spines," truncated ends, etc.)
and studied the fate of the peculiar features during subsequent fissions.
In many cases the abnormality gradually disappeared — the normal form
being gradually regained in a few generations. Sometimes, however,
the animals or their descendants became still more monstrous, and
finally died. No permanently abnormal races were ever obtained by
selecting monstrous individuals.

' Attention was first called, I believe, to ciliate monsters by Tatem (1870) wbo described
two specimens of Chilodon and one of Trachelius in which th'e "lip" was abnormally pro-
longed. His comment is worth quoting : "Malformations such as those I have cited have,
in my opinion, avalue beyond that of mere curiosities. ..for may theynot help to determine
the fixity or otherwise of a species through aberrant forms ? and thus a better knowledge
of what is to be regarded as essentially specific be ultimately arrived at." Curiously
enough, another "monster" described by Tatem is now recognised as a distinct ppecies
(Vorticella monllata).

'' The species studied "had the characteristics usually attributed to Paramecium
caudatum. "

162 On the Geneiics of the Ciliate Protozoa

84. The behaviour of a monstrous character during fission may be
illustrated by the abnormal race a of Jennings, which is fully described
for 22 generations. An abnormally bent organism divided into a normal
posterior daughter-individual and an anterior possessing a dorsal "spine."
In subsequent generations this spine persisted, passing sometimes to
the anterior, sometimes to the posterior individual. If we write A and
P for anterior and posterior individuals respectively, the transmission
of the spine for the 21 generations observed may be represented thus :

APAPAPAPPPPAAAPAAPAAA.

The last spined animal died. All the sister-individuals were normal,
save at the second generation. Here the anterior individual had a
small posterior ventral " tooth," which persisted for two further genera-
tions, passing each time to the posterior individual, and gradually
becoming smaller. At the next division it disappeared completely,
two normal individuals being formed.

85. Jennings (1908) gives particulars of other abnormalities in
Paramecium and their behaviour during fission, with a long discussion
of his results. Special mention may be made of a curious " race " in
which the individuals showed a tendency to remain united instead of
separating comjjletely during fission. This race was " extinguished by
natural selection " in competition with free and more active organisms.

86. McClendon (1909) obtained some results like those of Jennings
(§ 84), but in a different manner. As a consequence of centrifugiiig
some Paramecia^ he obtained an abnormal individual which divided
into two daughter-individuals each possessing a " hom." One of these
divided for 7, the other for 5 generations — the horn persisting and
passing to one of the daughter-individuals each time, the other being
invariably normal. Finally the horned animals died. In position and
size the horns differed in different organisms. " After each division
the horn is in a different position, and we can predict the position of
the horn in each generation by drawing an imaginary line bisecting the
animal in the preceding generation transversely."

87. From the observations of Balbiani (§ 81), Simpson (§ 82),
Jennings (§ 84), and McClendon (§ 86), the following conclusions may,
I think, be drawn. Abnormal growths, however produced, in Para-
mecium may be mechanically handed on for a number of generations.
Whether they pass to the anterior or posterior product of division is
purely a matter of chance, depending upon the position which the

' The species was P. caiidatum.

C. DOBELL 163

structure happens to occupy on the parent at the moment of fission.
In some cases the abnormality disappears owing to remodelling during
successive generations : in other cases the abnormal forms die. Normal
sisters of abnormal forms show no tendency to beget correspondingly
abnormal individuals. Such teratological variations are therefore
negligible as factors in the production of new races.

88. Additional information about ciliate monsters will be found in
the following papers: Balbiani (1891) — double monsters in Stentor ;
Balbiani (1892, 1893) — various monsters resulting from mutilation of
Paramecium, Stentor, etc.; Simpson (1901) and Calkins (1904) —
multiple monsters in Paramecium; Prowazek (1904) — double monsters
produced by cutting Stentor, etc.; Prowazek (1904 a) — Stylonychia mon-
sters with multiple hinder ends, resulting from " degenerative hyper-
regeneration " ; Calkins (1911) and Peebles (1912) — multiple and other
monsters produced by cutting Paramecia. Hereditary behaviour of
abnormalities is hardly touched on in these papers. Yet an interesting
fact is several times reported' — namely, that remodelling when it does
not take place in a monster itself may occur in its offspring; so that
in certain cases at least monstrosity is a temporary manifestation — the
peculiarity of the individual and not of its race. This is in accord with
the conclusion reached in the preceding paragraph.

89. Before leaving the events of the asexual period, I would
mention an interesting observation by Fermor (1913). The authoress
finds that reorganization of the nuclear apparatus may occur in Stylo-
nychia during encystment — without any sexual manifestations. In the
encysted animal, the meganucleus degenerates and disappears. The
micronucleus then separates into two parts — one of the products subse-
quently forming the new meganuclei, the other forming the new micro-
nuclei. Thus the organism which emerges from the cyst has undergone
a nuclear reorganization comparable with that which accompanies
conjugation (§ 5). Conjugation was never observed in the race of
Stylonychia studied. I know of no other similar observation-.

' This occurred, for example, in one of the double Stentors described by Balbiani
(1891).

^ It is possible, however, that a similar nuclear reorganization may occur at times in
unencysted, asexually reproducing organisms. Hertwig (1889) believed that such a process
("parthenogenesis") occurred in Paramecium, and a similar suggestion of the occurrence
of " autogamy " in the same form has more recently been made by Woodruff (1908 a).
The matter requires fuller investigation. [Whilst this article is passing through the
press, an important announcement has been made by Woodruff and Erdmann ("Complete
periodic nuclear reorganization without cell fusion in a pedigreed race of Paramecium,"
Proc. Soc. exp. Biol, and Med., Vol. xi. No. 3, 1914, p. 73), The authors state that they

164 On the Genetics of the Ciliate Protozoa

B. The Sexual Period.

90. Turning our attention now to the sexual phase of ciliate life
we must consider the causes and effects' of conjugation. We may begin
with certain isreliminaries to the process — considering first " assortative
mating." This has been proved biometrically by Pearl (1907) to occur
in Paramecium caudatum. He found that conjugants are smaller^ less
variable, and more alike than non-conjugants (as regards size and form).
The likene.ss between a pair of conjugants is "not due to any local
environmental factor" but to " homogamy " — individuals tending to
pair with their likes, not at random. These observations have been
confirmed by Jennings (1911 a) in both P. caudatum and P. aurelia.
Watters (1912) reports similar conditions in Blepharisma.

91. That assortative mating occurs in ciliates generally has not
been proved, nor has it been tlemonstratetl that conjugants are always
smaller than non-conjugants^ or that they are usually of the same size.
Marked differences in the sizes of a conjugating pair have often been
noted — for example, by Mulsow (1913) in Stentor. He noticed that
conjugants are smaller than non-conjugants, but found that only about
half the pairs'* were composed of similar sized individuals. In other
cases one individual was smaller than its partner — the difference being
sometimes very considerable. Doflein (1907) says that " in Paramecium
putrinum almost half the pairs are composed of distinctly different
individuals." Indeed, he was so impressed with the dissimilarities
observable between two conjugating individuals in many .sj)ecies that
he enunciated a " working hypothesis " to account for them. The
frequent suggestion that the differences between the members of a
conjugating pair are sexual in nature — the larger being female, the
smaller male — is manifestly due to a misunderstanding of the nature
of a conjugant (§ 14). For my own part, I do not consider that

have been able to show that " the rhythms in the division rate of Paramecium are the
phj-siologieal expression of profound nnclear changes," which "involve the formation of a
complete new nuclear apparatus."..." This nuclear reorganization is evidently a normal
substitute for typical conjugation."]

' I use these elusive terms in their colloquial sense, without prejudice to any conception
of causality.

- Previously observed by Maupas, Gruber and others.

^ Maupas (1889) enumenites 10 species in which the conjugants are smaller than the
non-conjugants, and 7 in which they are of the same size.

^ About 3000 pairs were studied— but not biometrically.

C. DOBBLL 165

" homogamy " in Paramecium means much. It is merely an expression
of the fact that a population of sexually mature individuals is more
uniform in size than is a population of adults, adolescents, and
children.

92. Is there a "eugamic period," or period of "karyogamic maturity,"
in the life-cycle of a ciliate ? Many experiments could be quoted to
show that there is not. For instance. Calkins (1902) concludes:
" ' Karyogamic maturitj' ' does not signify much when fertile unions
occur with individuals' in the 350th, the 410th, the 4.67th, and .500th
generations of the same culture." And Jennings (1913) records an
experiment (No. 9) in which he studied two lines of P. awelia, both
derived from one original ancestor, but only one of which had been
allowed to conjugate. "Under the proper conditions both sets con-
jugate at the same time, in spite of the fact that one has conjugated
at least four times since the other." These and similar observations
(especially those on reconj ugation — § 120 infra) seem to indicate clearly
that a " eugamic period," as conceived by Maupas, does not exist°.

93. R. Hertwig (190.5) states that when Dileptus is starved^ two
rapidly succeeding divisions ("hunger divisions") take place, resulting
in the formation of four small individuals from each original individual.
This is said to occur also in Didinium (Prandtl, 1906) and Paramecium
(Kasanzetf, 1901). "Since Infusoria which have undergone hunger-
divisions proceed to conjugate, a causal connexion seems to exist
between the two phenomena. The hunger-divisions correspond in
this respect with the maturation divisions of multicellular organisms."
It is true that these " hunger-divisions " may account for the smaller
size of conjugants — when they are smaller: but the remainder of
Hertwig's argument seems based on a false analogy. For since the
conjugant is not a gamete but a sexual individual (§ 12), it cannot
properly be compared with a germ-cell. The phenomena in a ciliate .
which are homologous with the two meiotic divisions in a metazoon
are the micronuclear divisions preceding fertilization (§ 13).

' Paramecium caudatum.

" It is possible, however, that under normal conditions conjugation maj' occur with
fairly regular periodic frequency. Jennings (1910) describes a race of Paraimeium which,
under suitable conditions, will conjugate again 5 days after conjugation: in other races
there was an interval of "a year or more." Something like a "eugamic period "may
couceivably exist, therefore, though it seems very improbable that this is the "explanation"
of these facts.

' Hunger, it will be recalled, is a condition conducive to conjugation, according to
Maupas (§ 20).

166 On the Genetics of the Ciliate Protozoa

94. Hunger was believed by Maupas (§ 20) to induce conjugation
in cultures containing sexually mature individuals. The ert'ects of
hunger upon the organism have since been specially studied by
Wallengren (1901 a, — Paramecium and Colpidium) and Kasanzeff
(1901, — Paramecium). Wallengren finds that hunger produces de-
generative changes in the cytoplasm (vacuolation, etc.) and meganucleus
(fragmentation, etc.), but not in the micronucleus. Starved individuals
are of conspicuously smaller size. These observations are confirmed by
Calkins (1904). Kasanzeff finds that starvation leads to an increase in
the size of the meganucleus. And R. Hertwig (1899, 1902, 1905, etc.)
has been led by these and similar observations to formulate the following
raisoii d'etre of conjugation — based upon his hypothesis of the karyo-
plasmic ratio (§ 30) : In the course of normal functional activity of the
organism, or as a result of hunger, the meganucleus grows at the
expense of the cytoplasm, thus causing an increasing disproportion
in the mass-relations of the one to the other. This disproportion may
be compensated by a reorganization of the nuclear apparatus : and
conjugation therefore takes place in order to bring about this result.
Conjugation is thus regarded as a means of regulating the karyoplasmic
ratio. Some remarkable experiments inspired by this idea have since
been made.

95. It will be recalled that the meganucleus is relatively larger
in ciliates kept at a low temperature than in those kept at a high
temperature (§ 66). It therefore occurred to Prandtl (1906) that if
he were to subject organisms adapted to a low temperature suddenly
to a higher, they would find themselves in an abnormal condition in
which the meganucleus was too large. In other words, the condition
which normally leads to nuclear reorganization through conjugation
could be thus brought about experimentally. A sudden change of
this sort ought, therefore, to lead to conjugation. The experiment
was tried with Didinium and Dileptiis, and with successful results.
Conjugation took place at the higher temperature. It should be noted
that in these experiments temperature was not the only factor con-
cerned. For Prandtl supplied his animals with plentiful nourishment
at room temperature, and then starved them at the subsequent tem-
perature of 25° C. Similar experiments were made on the vorticellid
Carchesium' by Popoff (1908 ((), with a like successful result. He found
further that starvation coupled with lowered temperature favoured the
production of males : whereas coupled with raised temperature it led to

' Wrongly stated to have been Epixti/lis in an earlier paper (Popoff, 1907).

C. DOBBLL 167

the production of females \ In all his experiments, however, starvation
introduces a complication : so that after carefully studying the facts
which he, Prandtl, and Hertwig have so far recorded, I am still unable
to understand the real significance of these interesting observations.
They seem to me suggestive rather than demonstrative.

96. The method employed by Maupas (1889) for inducing ciliates
to conjugate consisted in the simple procedure of transferring some
individuals from a large stock culture to a small culture on a slide.
As soon as the food in the smaller culture was exhausted, conjugation
occurred — provided the animals were " sexually mature." Essentially
the same measure has been frequently used with success by other
workers (Hamburger, 1904 ; Calkins and Cull, 1907 ; Enriques, 1907 ;
Jennings, 1913; Calkins and Gregory, 1913; Woodruff, 1914; etc.).
Jennings (1913) describing his method says: "In the evening large
numbers of the animals [P. caudatum and P. cmrelia] were taken from
the large cultures and placed in watch glasses; early the following
morning they were usually beginning conjugation." He believes that
conjugation occurs " not as a result of starvation, but at the beginning
of a decline in the nutritive conditions, after a period of exceptional
richness that has induced rapid multiplication" (Jennings, 1910). Calkins
had earlier (1902) concluded that "hunger is not a pre-requisite for
union, it apparently prevents conjugation."

97. Enriques has long maintained that " the necessary and sufficient
conditions for conjugation are environmental conditions" (1909 a). He
states (1907) that Colpoda steini will conjugate only when the depth of
the culture is 2 — 3 mm.- — never in deeper cultures. He further states
that the liquid from a culture in which conjugation is taking place,
when added to a non-conjugating culture, brings about conjugations in
it : and reversely, liquid from a non-conjugating culture, added to a
conjugating culture, causes cessation of conjugation. He concludes
that the onset of conjugation "does not depend upon mysterious con-
ditions developing themselves in the infusoria, but the modifications of
the circumambient liquid play the chief part."

98. Pursuant of this train of thought, Enriques (1909) has per-
formed a number of experiments with Gryptochilum nigricans. He

' Popoff incorrectly (§ 14) calls males aud females " microgametes " and "macro-
gametes."

^ Compare in this connexion the much earlier experiments of Everts (1873) on
Vorticella. He believed that conjugation was caused by the drying up of the water
iiu which the animals lived.

1G8 On the Genetics of the Ciliate Protozoa

says that this ciliate will thrive in an infusion of hay in distilled water:
but in this medium conjugation cannot occur. Conjugation depends
upon the presence of salts. Addition of NaCl, NaBr, or Nal in certain
concentrations causes epidemics of conjugation — the efficacy of these
salts being in the order given. [That is, the order of efficacy corre-
sponds with the order of the halogens in the periodic sy.steni, and is
the reverse order of their toxicity.] The effects produced by CaClj and
FeoCls were surprising. One part (by weight) of either salt in one
million parts of medium was sufficient to inhibit conjugation: but
stronger doses (1 : 10,000) caused intense epidemics of conjugation.
Addition of iron to the cultures had the most pronounced effect.

99. Similar but more extensive experiments have been made with
Paramecium caudatum by Zweibaum (1912). Stripped of detail, his
results are as follows. If a pure line oi Paramecium is richly nourished
in hay infusion, it will continue to multiply for an indefinite period
without conjugating. If organisms are from time to time tested by
placing them in solutions free from hay but containing (1) salts' in
strong or (2) salts in medium concentrations or (3) no salts, then still
no conjugations result. If however, the richly nourished culture is
changed — by removing the hay — into a " hunger culture," and if this
is similarly tested after the lapse of a considerable time (5 — 6 weeks),
then the trials result thus: in (1) and (3) no conjugations; in (2)
abundant conjugations. In other words, the conditions necessary for
conjugation in Paramecium, are — plentiful feeding, followed by starva-
tion, followed by treatment with salts in medium concentration. Con-
jugation can occur at temperatures from 9° C. to 29° C. (optimum
20° — 23°). There is an optimum concentration for each salt — which
was determined — and successful results can be obtained by substituting
glucose for salts. The most effective salt was found to be AICI3, which
in concentrations of N/24000 to N/48000 gave "always almost complete
epidemics" of conjugation. As they stand, these experiments appear
to be conclusive, though it is difficult to reconcile them with other
observations. It is to be hoped that the work of Ernnques and Zweibaum
will soon be repeated by independent investigators.

100. Some additional evidence of the influence of external con-
ditions in causing conjugation is given by Baitsell (1912). He found
that two lines of Stylonychia, derived from the same original organism
but kept in different media, behaved differenti}' as regards conjugation.

' Many different salts were tried, their several effects being described at length and
enumerated in 36 tables of experiments.

C. DOBELL 169

The line bred in hay infusion refused to conjugate : whereas the parallel
line in beef extract conjugated. He concludes that " the determining
feature was the medium used." '' Conjugation is induced by external
conditions."

101. Jennings (IJ)IO) has come to the conclusion that "the con-
ditions determining conjugation differ greatly in different races of
Paramecium {aurelia or caudatuin). Some races conjugate frequently,
and under conditions readily supplied in experimentation. Others,
under the same conditions, conjugate very rarely or not at all."
Calkins and Gregory (1913) also share the view that there are con-
jugating and non-conjugating lines of Faramecmiii. Such a hypothesis
would help us to comprehend the extraordinarily different observations
made by different workers : but there is a very serious difficulty in the
way of accepting it — namely, we have absolutely no proof that any race
exists, which, under suitable conditions, is unable to conjugated Un-
doubtedly the weightiest evidence for the existence of such a race was
that furnished by Woodruff (§ 39), whose pedigree line of P. aurelia
consistently refused to conjugate for over 4000 generations. We
now hear, however, that after more than this number of generations,
conjugations have at last taken place (Woodruff, 1914). The likelihood
that anybody will ever succeed in demonstrating with any plausibility
that any given race of ciliates is incapable of conjugating, seems there-
fore immeasurably remote.

102. We may now pass to a consideration of the effects of con-
iugation. We have already seen that Maupias considered the chief
result of conjugation to be " karyogamic rejuvenation " (§ 17). His
work has frequently been misinterpreted as showing that conjugation
reinvigorates the stock in which it occurs — that after a number of
asexual generations the stock becomes weakened with age and divides
more slowly, but may be restored to its former vigour and rate of
reproduction by means of conjugation^. The work of Maupas does
not show this. Calkins (1904), however, has adopted this standpoint,
concluding from his work "that conjugation does actually rejuvenate
and overcome the conditions of so-called ' old age '." He appears to be

• I do not, of course, herebj' controvert the general statement of Jennings (1910) that
"the conditions for conjugation are different in different races."

'■' This view was first definitely advocated by Biitschli (1875, 1876) who believed that
conjugation resulted in "eine erhohte Teilungsfahigkeit," which he interpreted as a sign
that "Verjiingung" had been brought about. Balbiani's views were — for a time, at least
— similar.

170 On the Genetics of the Oiliate Protozoa

still of the same opinion {vide Calkins and Gregdry, lOK^), and many
others have at various times concurred.

103. Yet R. Hertwig (1889) clearly showed that such a conclusion
is not justified. He found that the rate of fission is not diminished
before conjugation, but rather increased. He performed the ingenious
experiment of forcibly separating a pair of prospective conjugants soon
after they had united, and cultivating them further. Far from dying,
they continued to divide noi-mally for many generations — thus showing
that they were not incapable of further multiplication, or in need of
" rejuvenation " through conjugation. Hertwig's conclusion was the
opposite to that generally drawn (§ 102). He believed that the rate
of fission becomes abnormally high before conjugation, and that the
sexual act has the effect of diminishing and normalizing it.

104. Hertwig's experiment has been repeated by Calkins (1902),
and on a large scale by Jennings (1913). The latter has dealt with
the facts exhaustively, and his conclusion can hardly be disputed. He
writes : " In view of the large number of experiments made by Maupas
on this point, the absolute agreement of his results with those of
Richard Hertwig ; the fact that these men are perhaps the most
thorough investigators that have ever worked along these lines ; the
further fact that there exist no careful experimental results opposed
to these ; and finally, the very large body of evidence presented in the
present paper^ all giving the same results — is it not time that the
statements or implications that in the infusoria conjugation results in
increased reproduction should disappear from the literature of .science ? "
The answer is emphatically affirmative. And it should be noted that
it sweeps away all arguments that conjugation causes "rejuvenation,"
" increased vitality," and the like : for this " vitality " itself is ultimately
measured by the rate of fission.

105. The effects of conjugation have been studied in elaborate
detail by Jennings (see especially Jemiings 1911a, 1913: Jennings
and Lashley, 1913, 1913 a). The extensive nature of his researche.s,
the large number of details which he has endeavoured to elucidate, the
vast array of facts which he has recorded — all these make his work
excessively difficult to understand or to summarize. It can be appre-
ciated in the original only. Comparison of experiment with experiment,
conclusion with conclusion, leaves me — after devoting much time and
attention to the matter — still in doubt as to what all this work really
amounts to. Analysis in detail is here impossible, and I must be

' Jennings (1913).

C. Do BELL 171

content with the baldest statements and criticisms of Jennings's general
conclusions. It may be said at once that the programme of his work is
admirable. He has studied both "wild" and pure strains of Furamecium
caudatum and P. aurelia, by means of observation and experiment and
where possible by biometric methods. Certain characters (fission rate,
size, etc.) were studied in individuals or their progeny belonging to
the four classes (a) non-conjugants, (6) conjugants, (c) exconj ugants,
(d) "split" or "unpaired" conjugants — i.e. individuals forcibly separated,
and bred further, after they had united for conjugation \ Knowledge
of the behaviour of all these classes of individuals should obviously give
definite information concerning the effects of conjugation.

106. The first general conclusion to which Jennings comes is that
conjugation causes variation'^ — " Conjugation produces within a pure
race heritable differentiations ; so that as a result races diverse in their
heritable characters arise from a single race with uniform heritable
characters." This conclusion is drawn — speaking generally — fi-om the
demonstration that, in a given race, the progeny of non-conjugants and
split conjugants are alike, but differ from the progeny of exconjugants.
There is greater variability among the progeny of the last. Since
conjugation is the only known factor which differentially affects the
two groups, it seems justifiable to conclude that it is in some way a
" cause " of variation. Yet this conclusion is remarkable. We have
seen that non-conjugating "pure lines" are constant in character — the
differences which the constituent individuals display being temporary
and not heritable (§ 65). We have seen further (§ 90) that within the
pure line " assortative mating " occurs, so that the members of a con-
jugating pair of organisms are more alike than those of a non-conjugating
pair selected at random. And yet after conjugation these like individuals
produce progeny which are unlike themselves and their race.

107. Jennings's second conclusion is at first sight even more strange.
It is that " conjugation results in biparental inheritance." (See Jennings
and Lashley, 1913, 1913 a.) The meaning of this misleading expression^
will be clear from the evidence upon which the conclusion rests. We
have seen (| 106) that the progeny of non-conjugants and split con-
jugants, within a pure race, are alike : and that the progeny of conjugants

1 E. Hertwig's experiment {§ 103).

'' This, of course, is a doctrine long ago promulgated by Weismann.

' I say "misleading" although Jennings explains the term (Jennings and Lashley,
1913, p. 457): for nothing is here "inherited" from the "two parents" save the power
to differ from them in a given respect.

172 On the Genetics of the Ciliate Protozoa

tend to be different. But biometric proof is now offered that the progeny
of one member of a pair of conjugants tend to be like the progeny of
the other, owing to the hereditary influence of both " parents " (con-
jugants) on all the progeny. It thus appears that conjugation simul-
taneously produces uniformity and diversity. I take this paradox to
mean that if two similar individuals of the same race conjugate, then
the ])rogeny of both will ditt'iT fniiii the original race, though the
progeny of one will resemble the progeny of the other in whatever
respects it diff'ers from the original race. Or, in other terms, a pair
of conjugants a, and a„, belonging to a race a, produce after conjugation
progeny forming races bi and h., — differing from a, but resembling one
another in both being b.

lOS. Both the fundamental conclusions reached by Jennings appear
to me unproved. My chief difficulty is that I cannot find convincing
evidence of a single concrete instance in which, from a known race —
constant in a certain character — a new race — permanently diverse in
this character — has arisen as a result of conjugation. As an abstract
biometric proposition, it is no doubt demonstrated "that conjugation
among the members of a pure race does result in differentiations that
are inherited," so that from a uniform race diverse races might seem to
arise. Now the diversity of the new races is manifested mainly in
(1) mortality, (2) size, (3) rate of fission. In the first case, Jennings
(like others) has found that many exconjugants, or their immediate
descendants, die. The consequence of conjugation in these cases is
really death. It is true there is increased variation in mortality among
the progeny, but the individuals which manifest this diversity merely
die out, so that no new races are produced in this fashion. Concerning
the second character — size — the evidence' seems to me unsatisfactory.
All that it appears to show is that although conjugants are smaller
than non-conjugants of the same race, their progeny are not : and that
there is greater variability in the size of individuals formed by fission
from exconjugants during the first few days after conjugation, than is
seen in the progeny of ordinary non-conjugants. Definite evidence of
the production of a new race of permanently different size — as a result
of conjugation — I can nowhere find. Perhaps the best is that which
can be extracted from the paper of Jemiings and Lashley (191:3 a), from
which it can be inferred that the descendants of 43 pairs of exconjugants,

' .Jennings (1911a). Further evidence is given in 1913 (expt. 9), but of this .Jennings
himself says "the results there given are by no means conclusive, the matter requires
further study."

C. DOBBLL 173

measured 25 days after conjugation, all possessed a smaller mean size
than that of the race from which they were derived. The number of
individuals measured is not great, however, and one may doubt whether
permanently smaller races^ had been produced as a result of conjugation.

109. Concerning the third character — rate of fission — the evidence
appears at first sight conclusive. The biometric results show clearly
that there is a difference (greater variability) in the fission rate of the
descendants of exconjugants as compared with those of non-conjugants
or split conj ugants — all of the same pure line. But as Jennings himself
points out " conjugation increases the variation mainly toward the lower
extremity of the range" — that is, the effect of conjugation is to retard
the rate of fission. Is not this merely another aspect of the same
condition which is otherwise manifested as " high mortality " and " loss
of vigour" after conjugation? Jennings's "Experiment 6" seems to
me the key to the matter. " This exjDeriment as a whole shows the
fact that after conjugation the organisms are in a condition such that
many may die, while those that have not conjugated live ; and the
further fact that the rate of reproduction is made slower by conjugation,
remaining in this condition for about two months," after which it " has
regained about the usual rate." If the result of this experiment may
be regarded as typical, then it indicates that the lowering in fission rate
following conjugation is transient, recovery occurring sooner or later.
It is demonstrated that after conjugation the organism and its progeny
are weaker, or less resistant to external conditions (shown by higher
mortality, lagging fission rate, unstable size, abnormalities, etc.) for a
certain time ; and that complete recovery to the normal state preceding
conjugation occurs subsequently : but I find no proof that from a race
with a given fission rate, another race with a permanently different
fission rate has arisen as a result of conjugation'-.

110. Turning now to Jennings's second proposition (§ 107), I can
make criticisms of only a very general nature. I am not able to judge
of the validity of the biometric methods employed for its demonstration.
It appears to me, however, that to prove " biparental inheritance," the

' The fact that all the races are smaller is significant. One can hardly suppose that
conjugation in this race always leads to a permanent reduction of size. It seems much
more likely that there was some environmental factor (e.g. food or temperature — cf. §§ 60,
66 et seq.) affecting all the cultures alike.

2 A similar criticism to the above has already been made by JoUos (1913 a). It seems
to me unfortunate that Jennings should have selected for his chief study a physiological
character— fission rate — which is so greatly influenced by environmental conditions (§ .51
et seq.).

Journ. of Gen. iv 12

174 On the Genetics of the Ciliate Protozoa

descendants of a pair of exconjugants must form two fairly homogeneous
lots — in order that any comparison between them is possible. By taking
the mean for some lines descended from each exconjugant, such a homo-
geneity may be introduced when it does not really exist. For if the
progeny of an exconjugant differ from one another — the differences
being, urgumenti causa, caused by conjugation — how can any real
comparison be made between all the different organisms which could
be obtained from one exconjugant — the number is unlimited — and
those from its partner ? It is physically impossible to study the rate
of fission in more than a few lines descended from an exconjugant, for
at each fission the number of possible lines to be studied is multiplied
by two. Granted that conjugation causes variation, so that the lines
derived from different progeny of one individual exconjugant display
different fission rates, how is it possible to reach any definite conclusion
by studying certain arbitrarily selected lines representing only an in-
finitesimal fraction of all possible lines ? It seems to me that if
conjugation gives rise to variations of this sort — as certain experiments
seem to show — then any real demonstration of " biparentaj inheritance "
in fission rate is impossible. I cannot comprehend how Jennings's
results in this connexion can have any value beyond suggestiveness.
111. Calkins and Gregory (1913) have recently published an
account of the variations observable in different lines of Paramecium
derived from a single exconjugant. In one case as many as 30 lines
from one exconjugant were studied — that is, a single line from each of
30 out of the 32 individuals formed by the first five fissions after con-
jugation. The authors conclude: "The results of this study show that
physiological and morphological variations in the progeny of a single
exconjugant of Paramecium caudatum are fully as extensive as the
variations between progenies from different exconjugants. The argu-
ments based upon the latter variations to the effect that conjugation
is for the purpose of originating variations cannot therefore be sustained."
The authors appear to believe that they have invalidated Jennings's
general conclusion that conjugation causes variation. If so, their
argument is a palpable non sequitur. What they have shown is that
the progeny of an exconjugant differ from one another — that there is
considerable variability among them. Whether the variability is the
effect of conjugation or not, there is no means of judging: for no
comparative study of non-conjugants and split conjugants of the same
race appears to have been made — a study which is es.sential if the effects
of conjugation are at issue. The real importance of these observations

C. DOBELL 175

seems to me to lie in the fact that they afford additional grounds for
doubting the validity of Jennings's conclusions concerning " biparental
inheritance "(§ 110). It maybe noted further that some evidence is
given that the differences between the surviving lines derived from the
same exconjugant tend to disappear in the course of time — a tendency
which is of interest in view of what has previously been remarked (§ 109).

112. It is noteworthy that Pearl's (1907) biometric study of
Paramecium led him to a conclusion diametrically opposed to that
of Jennings (§ 106). He says: "There is no evidence that conjugation
tends to produce increased variability in exconjugants. All the evidence
indicates, on the contrary, that conjugation serves... to preserve relative
stability of type." The same standpoint is taken up by Enriques (1907),
though chiefly on a priori grounds.

113. A curious observation bearing on the effect of conjugation
has been made by Jollos (1913). He states that his arsenic-resistant
race of P. caudatum (§ 71) has lost its resistance as a result of conjuga-
tion\ Progeny of exconjugants displayed merely ordinary resistance,
whereas progeny of non-conjugants (a pai-allel line) continued to display
their acquired increased resistance for a long period. If this observation
is substantiated and confirmed, it appears to indicate that conjugation
is a barrier to the transmission of an acquired physiological character —
that it eliminates rather than originates variation.

114. It was believed by Calkins (1902) and Cull (1907) that the
effects of conjugation are not the same on the two members of a pair
of exconjugants — the progeny of one tending to survive, of the other
to die out. This was interpreted as evidence of " incipient fertilization "
in Paramecium — the form studied. When the nature of a conjugant
is properly understood, however, it is clear that this is based on a con-
fusion of ideas. The conjugant is neither a male nor a female, nor
a gamete of any category (§12 et seq.). To speak of " incipient fertiliza-
tion " or " incipient sexuality- " in this connexion is meaningless. It is
of interest, however, to know whether there is really a difference in the
fate of the progenies of a pair of conjugants, and the matter has been
exhaustively studied by Jennings and Lashley (1913). They conclude
that "if one member of a pair survives, the other member tends to
survive also ; if one dies out the other tends to die out also." The

' After conjugation the resistance is said to disappear "mit einem Schlage": but it is
also stated that the progeny of the exconjugants were not tested until two weeks after
conjugation, so that the "suddenness" of the loss is hardly demonstrated.

- Jennings and Lashley (1913).

12—2

1 76 On the Genetics of the Ciliate Protozoa

effects of conjugation tend to be the same — not different — for both
members of a pair. This deimmstration furnishes the chief evidence
for " biparental inheritance" in I'amineciuiii^ (§107).

115. Enriques (1908) states that in CliUodim, the two conjugants
are alike at the beginning of conjugation: but during conjugation they
become differentiated into a longer and a shorter. This phenomenon
is called "hemisex" — Enriques regarding the longer conjugant' as "half
female," the shorter as "half male." What the significance of this
"se.Kual differentiation as an effect of conjugation" may be, is obscure.
It is quite certain, however, that the phenomenon has nothing to do
with sexual differentiation properly so called. For from the point of view
of sex, the conjugants are identical — both being hermaphrodite (§ 14).
It may be noted that Jcimings (1911 «) has observed a tendency to
" equalization " during conjugation in Paramecium. The two members
of a pair of conjugants tend to become more alike — not less alike —
during the process.

116. We have seen (| 20) that Maupas regarded "inbreeding" or
" interconjugation^" as productive of harmful consequences. Some
observations bearing upon this matter have since been made, though
they are recorded for the most part incidentally. Cases of apparently
successful conjugation between closely related individuals are recorded
by Joukowsky (1898), Calkins (190'2), Jennings (1913)-' and others.
In other cases, however, such conjugations ajjpear to have been harmful.
Baitsell (1912), for example, found that the descendants of a Stylonychia
interconjugated readily: but all the exconjugants dietl. Calkins (1912)
made similar observations on closely related individuals of Blepharisma :
"conjugation is equivalent to a death warrant." Nevertheless, if inter-
conjugation really has harmful effects, it is impossible to reconcile this
with the facts (1) that conjugation occurs normally between individuals
belonging to the same pure line — not between those of different lines

' It is important to notice what the characters are which are "biparentally inherited"
— namely death, or survival. The characters are not racial characters — save in so far as
" survival " is a character of every living race.

- Enriques incorrectly — and inconsistently — calls the conjugants "gametes." When
he speaks of an organism as being "half male," I do not understand what he considers
the other half to be. The term " hemisex " seems to me ambiguous — or else incorrect.

' I use this term to denote conjugation between closely related organisms — i.e. between
descendants of the same ancestor. The term "inbreeding" is equivocal, and " intercon-
jugatiou " seems to me a more suitable word. Conjugation is not an act of reproduction
(§ 1.5), and therefore there is really no breeding in the process.

* In Paramecium putrinum, P. caudatum, and P. aurelia respectively.

C. DOBBLL 177

(Jennings, 1911 a) ; and (2) that homogamy occurs in addition (Jennings,
1911a). The inference ft-om these two facts seems clearly to be that
" inbreeding " is the rule — at least in Paramecium.

117. An extreme case of interconj ligation has been recorded by
Jennings'. He obtained no less than nine successive interconjugations
in the descendants of the same individual. " The progenitor of the race
vfas a single individual ; its progeny conjugated among themselves ; from
these conjugants a single exconjugant was taken and allowed to multiply
till there was conjugation among these." An exconjugant was again
isolated and allowed to multiply — and so on, nine times in succession
(in the complete experiment). It appears — though Jennings does not
emjjhasize the fact in this connexion — that the mortality among the
final descendants was excessively high.

118. A case of conjugation between very closely related individuals
is recorded for the colonial Vorticellid Opercularia by Euriques (1907).
He states that male and female conjugants (§§ 6, 14) are formed by an
original " indifferent " individual'- — incapable of conjugating — dividing
into a large and a small product, the latter dividing again into two
smaller individuals. The large individual becomes a female conjugant,
the two small individuals males. It is stated that conjugation may
occur between males and females formed in this manner from the same
indifferent individual''. Calkins (1912) has even recorded a case of
conjugation in Blepharisma in which the two conjugants were the
products of fission of the same individual — " the closest case of paedo-
gamy in ciliated protozoa on record." Death followed conjugation — as
in all eases observed in Blepharisma.

119. The general conclusion to be drawn from the recorded cases
of interconj ugation is by no means clear. It is evident that closely
related individuals will conjugate readily with one another : but the
ultimate effect of such conjugation on the jjrogeny is not evident,

1 Jennings (1913, expt. 13). Jennings speaks of this interconjugation as " self-
fertilization — which it certainly is not. He obtained eight successive interconjugations —
" to avoid, so far as possible, the heterozygotic condition "' — and then studied the effects
of conjugation at the ninth. Since the general conclusion from this was that conjugation
"increases greatly the variability," I cannot understand how previous conjugations are
supposed to eliminate "the heterozygotic condition." The mathematical treatment of
"self-fertilization" seems to have no bearing on the actual phenomena concerned.

'^ It may be noted that Enriques (1907) states that in the Vorticellid Carclieshini, the
branches of the colony are differentiated as males, females, and " indiflerents." This was
not confirmed by Popoiif (1908 «).

^ I do not know how it was possible to make this extremely difficult observation with
any certainty.

178 On the Genetics of the Ciliafe Protozoa

because the effects of conjugation with uurehited individuals have never
been studied simultaneously.

120. This seems the proper place at which to mention the i)heno-
nienon of "reconjugation" discovered by Enriques (1908). He found
that exconjugants of Chilodon, instead of dividing, sometimes proceed
to conjugate again — either with other exconjugants or with ordinary
conjugants. The same thing has been recently observed in Paramecium
by Klitzke (1914). Conjugation may be effected by an exconjugant or
by the products of its first fission. These observations clearly show
that the stimulus to conjugation — whatever it may be — may exist
without a series of asexual fissions having intervened since a preceding
conjugation. The fate of the progeny of " ex-reconjugants" has not
been adequately described.

121. I may here mention some cases of what may be called "mis-
conjugation." Doflein (1S)()7) says that if conjugating Parainecia are
forcibly separated, they will reunite with other individuals — either
non-conjugants or conjugants of a different staged Abnormal con-
jugations may thus be brought about, the results of which are not
recorded. Many observers have reported abnormal conjugations of
three or more individuals-. Several such unions of three individuals
were observed by Mulsow (1913) in Stentor: but to my knowledge no
investigator has yet ascertained the consequences of these misconjuga-
tions. Doflein (1907) further describes, in Paramecium piitrinum and
Styloiiychia mytilus, "agamic fusions" of two individuals, "cytoplasm
with cytoplasm, nuclei with nuclei, so that an apparently quite normal,
but relatively very large individual resulted." The subsequent be-
haviour is not described'.

122. In conclusion, a word may be said about "hybridization" in
ciliates — I mean cross-conjugation between two individuals of different
specie-s. I know of but a single case in which this is alleged to have
happened. Apart from this, there appears to be no recorded case of
"crossing" even between individuals belonging to different pure lines

' Doflein gives an illustration of this, which, according to Klitzke (1914), really depicts
a conjugation between an ordinary eonjugaut and a "reconjugant" (§ 120). This inter-
pretation certainly appears plausible.

- These are described, for instance, by Stein, Eugelmann, Jickeli, Gruber, Plate and
Maupas — among the older observers — and generally in Stylonychia or P. putrinum.

' But Engelmann said that these compound organisms {Stylonychia) can grow and
multiply — a statement as yet unconfirmed. Maupas believed that such fusions have
nothing to do with conjugation — the fused animals being monstrous, and their divisions
irregular and incomplete.

C. DOBELL 179

of the same species'. The case to which I refer is given by Simpson
(1901), who states that twice (out of 21 attempts) he succeeded in
getting conjugations between Paramecium caudatum and P. aurelia.
"After separation each of the exconjugants divided once: on the third
day they died off." The account is extremely unconvincing, and I think
it is infinitely more probable that Simpson was deceived than that
cross-conj ugation occurred.

CHAPTER IV.

General Conclusions.

123. In this final chapter I propose to consider very briefly certain
results of the genetic study of the Ciliata. I would point out that this
chapter is not a summary of previous chapter.s ; nor is it intended to be
a substitute for them — to enable the reader to dispense with the facts
there set forth. Each of the preceding chapters is itself a series of very
brief and incomplete summaries, which form — in part — the premisses
from which the following conclusions are drawn. If my conclusions
appear absurd and wrong, they may nevertheless incite further inquiry
into the evidence upon which they rest. This is my desire.

124. It is quite clear to me — and I have every confidence that
sooner or later it will be equally clear to others — that many of the
problems now associated with the ciliates do not exist in nature. They
are really dialectic — not problems of concrete biology. They are off-
shoots of the fallacies involved in the " cell theory," and of the unbridled
academic speculations concerning evolution which were fashionable at
the end of last century.

125. The fundamental error in the conceptions of Maupas and his
followers is due to the "cell theory." The ciliate has been called a
" cell," and certain constituent elements of the metazoan body have
been given the same name. Consequently it has been assumed that
an exconjugant is the homologue of a fertilized metazoan ovum ; and
that the whole succession of its descendants is homologous with the
entire body of a multicellular animal^. It has thus become possible

' "Assortative mating has clearly the effect of keeping differentiated races from
mixing. ...When a culture containing two species conjugates, the two as a result of the
assortative mating remain quite distinct" (Jennings, 1911a).

- This view has been advocated most strongly in recent times by Calkins (1909). The
reader who wishes for a fuller analysis of the matters here merely touched upon, will find
it in an earlier publication (Dobell, 1911).

180 On the Genetics of the CUktte Protozoa

— and even necessaiy — to look for periods of adolescence, maturity,
senescence, and death, in the successive generations of individiuils
derived from an exconjugant. But as soon as it is realized that a
ciliate is a non-cellular but complete organism, homologous with
a whole metazoan ; as soon as it is realized that the only resemblance
between a ciliate and a metazoan cell was created by biologists when
they gave the name " cell " to both : then it will also be realized that
there is no reason to expect that successive generations of ciliate
individuals will manifest the same series of phenomena as is manifested
by a single individual metazoon during its life-time. One of the chief
results of the researches recorded in previous chapters is the demonstra-
tion that events, whose occurrence we have absolutely no reason to
expect, do not, in fact, occur.

126. It has been shown (§ 48) beyond all reasonable doubt that
under suitable conditions ciliates are able to live and multiply, in their
own fashion, for an unlimited time' — like all other organisms that are
well adapted to their environment. To ask whether they become
"senile" m the course of successive generations, whether "protoplasmic
old age " sets in, whether " rejuvenation by fertilization " is a periodic
necessity, and so on — to ask such (juestions is to propound problems
which are either unanswerable or meaningless. Among ciliates, off-
spring are formed by the division, growth and differentiation of the
protoplasm of their parents — as in all other organisms. If "immortality "
can be predicated of the ciliates, it can also be predicated of all other
organisms in the same sense. "Are the descendants of a ciliate older
than their progenitor ? " is the same question as " Is the child older
than its father?" There is no jjroblem here at all, for the answer is
self-evident as soon as the (juestioner intimates what he means by "old."

127. That conjugation is able to "rejuvenate" is a belief whose
origin is revealed in the word " fertilization." Conjugation is accom-
panied by " fertilization," and has consequently become confounded with
the original connotation of that term". Probably no living biologist
would care to advocate the view that the cytological phenomenon now
called " fertilization " is a process of " revitalizing the germ " — though
more than one can be found to discuss whether "conjugation results in
rejuvenescence." But quite apart from any mental or verbal tangles

1 I believe there are no a priuri reasons, or arguments from analogy with other non-
cellular organisms, which indicate that the ciliates arc incapable of continuing to multiply
asexually— in favourable circumstances— for an unlimited number of generations.

^ This was, of course, pointed out long ago by Weismann.

C. DOBELL 181

such as this, it may be stated now with considerable confidence, as a
concrete proposition, that conjugation in the ciliates does not result in
rejuvenation — no matter whether a literal or metaphorical meaning be
attached to the word.

128. Experimental inquiries into the factors which determine division
and hereditary transmission of characters ; into the factors which deter-
mine or inhibit conjugation ; into the results which conjugation itself
determines, whether in originating or eliminating variations ; and into
the origin of variations, — all these investigations have yielded a great
mass of facts of great interest and suggestiveness. But these facts —
in my opinion — throw little light upon the great central problems
concerned. Rather do they throw into vivid relief the formidable
complexity of all biological problems as presented by the Protozoa.
It is safe to prophesy that when the known facts have been doubled
or trebled, the ironical statement — which now prefaces so many memoirs
on the ciliates — that " the Protozoa are the simplest organisms in which
to study the great problems of biology " will disappear from biological
literature.

129. The tradition which finds expression in such statements as
this is at present almost universally received. It is believed that the
non-cellular organisms display vital phenomena in a more elementary
and therefore more easily comprehensible form than other organisms.
From his earlier physiological studies Jennings (1906) rightly con-
cluded that " the behaviour of the Protozoa appears to be no more
and no less machine-like than that of the Metazoa." " Action is as
spontaneous in the Protozoa as in man." This truth should never be
forgotten. It is vain to seek for simple mechanical factors which
" induce conjugation " in ciliates. For conjugation is the resultant
of many external and internal factors — environmental opportunities,
inherent inclinations and potencies — which are no less complex and
no more easily comprehensible than the factors which result in com-
parable phenomena in man. They are really less easy to understand,
because we have no conception of what the " motives " may be which
actuate a brainless, non-cellular creature.

130. In the foregoing pages I have confined myself to recording
and analysing facts and concrete questions. It is often asked " What
is the significance, or meaning, of conjugation?" This question is
itself meaningless. As well might one inquire the meaning of the
moon. I have purposely avoided all such questions, though they have
been freely debated by others. I have also avoided all discussion of

12—5

182 On the Genetics of the Ciliafe Protozoa

teleological interpretations of conjugation and other phenomena.
Whether conjugation is for the purpose of originating variations, or
for any other purpose, is to my mind an idle discussion : and I have little
liking for the "explanations" which have been given of the phenomenon
— explanations which seem to me to explain nothing and lead nowhere'.
So many real problems which may be attacked by means of experiment
are still unsolved, that I think the discussion of more remote problems
may be profitably postponed.

131. No new light has been thrown upon the great problems of
organic evolution by a study of the ciliates. They have revealed no
real indication of the manner in which evolution has proceeded, or is
proceeding, within the group": a fortiori, they tell us nothing of the
process of evolution in general. The facts so far determined could,
indeed, be used with far greater force to support the doctrine of the
fixity of species. Moreover, even if it were possible to draw any
general conclusions from the ciliates themselves, it would not be
justifiable as yet to extend them even to the other groups of Protozoal
The ciliates are so curiously organized that in many ways they stand
alone among animals. What is true of ciliates is not necessarily — or
even probably — true of most other organisms. These will be unwelcome
sayings to many, but I believe they are true. And all will admit that
it is better to face the facts, however distasteful and uncontbrmable to
theory they may be, than to veil them with that former assurance con-
cerning organic evolution which, as is fast becoming evident, was chiefly
begotten of ignorance.

' Compare for example ihe following: "Conjugation should be regarded as a set of
phjsico-chemical phenomena resulting in a sort of cellular purification" (Loisel, 1903).
"Conjugation appears to us as above all a set of chemical phenomena which counteracts
another set of chemical phenomena — .senescence" (Loiael, 1903 b), etc.

^ I leave out of account the numerous fanciful speculations concerning the phylogeny
of the ciliates which, I know, afford satisfaction to many. I am here considering the facts
relating to the ciliates on their merits — apart from any preconceived interpretations.

^ I believe there are few prutozoologists who would eudorse the opinion of Calkins
(1909) that Paramecium is "a typical protozoou."

Imperial College of Science, London.
March, 1914.

C. DOBELL 183

ANALYSIS OF CONTENTS.

N.B. 7%e numbers refer to paragraphs.

Preface.

Chapter I. Organization and Life of a Ciliate 1 — 1.5. , "

Structure 1 — "2.

Eeproduction 3.

Encystation 4.

Conjugation .5 — 8.
Meiosis 7.

Ciliate compared with Metazoon 9 — 15.
Chapter II. Life-cycle acCoirding to Maupas 16 — 22.

Life-cycle 17—18.

Division 19.

Conditions for conjugation 20.

Senescence 21—22.

Chapter III. Results of later investigators 23—122.

A. Asexual period 24 — 89.

Duration of period; "senescence," " depression," — causes and effects 24 — 44.
Mechanics of division 45 — 50.

Effects of chemical and physical agents on division and growth 51 — 60.
Variation and inheritance 61 — 88.

Normal inheritance 61.

Pure lines 62—65.

Production and inheritance of variations 66 — 88.
Monsters 81—88.
Nuclear reorganization without conjugation 89.

B. Sexual period 90—122.

Conjugation 90 — 115.

Assortative mating 90—91.
"Karyogamic maturity" 92.
"Hunger divisions" 93.
"Causes" of conjugation 94—101.
Internal 94—96.
External 97—101.
"Non-conjugating races" 101.
" Effects " of conjugation 102 — 115.
" Bejuvenescence " 102 — 104.
Efl'ects on variation 105 — 115.
"Biparental inheritance," 107, 110—111, 114.
" Hemisex " 115.
Interconjugation ("inbreeding") 116 — 119.
Eeconjugatiou 120.
Misconjugation 121.

Cross-conjugation ("hybridization") 122. ,

■Chapter IV. General conclusions 123 — 131.
Analysis of Contents.
.Bibliography.

184 0)) the Genetics of the Ciliate Protozoa

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0. DOBELL 185

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186 On the Genetics of the Ciliate Protozoa

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C. DOBBLL 187

Jennings, H. S. (1910.) "What conditions induce conjugation in Paramecium V

{Journ. exp. Zool. Vol. I.x. p. 279.) 92 n., 96, 101, 101 n.
. (1911.) " Pure lines in the study of genetics in lower organisms." {American

Nat. Vol. XLV. p. 79.) 63, 65.
. (1911a.) "Assortative mating, variability and inheritance of size, in the

conjugation of Paramecium." {Journ. exp. Zool. Vol. xi. p. 1.) 90, 105, 108 n.,

115, 116, 122 n.
. (1912.) "Age, death and conjugation in the light of work on lower

organisms." {Pop. Sci. Monthly, June, p. 563.) [Summary and discussion.]
. (1913). "The eflect of conjugation in Paramecium." {Journ. exp. Zool.

Vol. XIV. p. 279.) 92, 96, 104, 104 n., 105—109, 116.

and Haegitt, G. T. (1910.) "Characteristics of the diverse races of Para-
mecium:' {Journ. Morpli. Vol. xxi. p. 495.) 63, 65, 73 n., 78.

and Lashley, K. S. (1913.) " Biparental inheritance and the question of

sexuality in Paramecium." {Journ. exp. Zool. Vol. xiv. p. 393.) 105, 107,
107 n., 114, 114 n.

. (1913a.) "Biparental inheritance of .size in Paramecium." {Ibid.

Vol. XV. p. 193.) 10.5, 107, 108.

JoLLOS, V. (1913.) " Experimentelle Untersuchungen an Infusorien. (Vorl.

Mitteil.)" {Biol. Centralbl. Bd. xxxui. p. 222.) 55, 56 n., 69, 70, 71, 113.
. (1913 0!.) "Ueber die Bedeutung der Conjugation bei Infusorien, usw."

{Arch. f. Protistenk. Bd. xxx. p. 328.) 109 n.
JoCKOWSKY, D. (1898.) " Beitriige zur Frage nach den Bedingungen der Vermehrung

und des Eintritts der Konjugation bei den Ciliaten." ( Verhandl. nalurhist.-med.

Ver. Heidelberg, Bd. vi. (n. F.), p. 17.) 25, 55, 57, 60, 116.
Kasanzeff, AV. (1901.) "Experimentelle Untersuchungen iiber Paramecium

caudatum." (Inaug.-Diss. Zurich. Summarized by Piitter, Zeitschr. allg.

Physiol. Bd. 1, 1902, p. 251.) 79, 93, 94.
Klitzke, M. (1914.) "Ueber Wiederconjuganten bei Paramecium caudatum."

{Arch. f. Protistenk. Bd. xxxill. p. 1.) 120, 121 n.
KOLAGIN, N. (1899.) "Zur Biologie der Infusorien." {Le Physiologiste Russe,

T. I. p. 269.) 26.
Le Dantec, F. (1897.) "La regeneration du micronucleus chez quelques In-

fusoires cilies." {C. R. Acad. Sci. Paris, T. cxxv. p. 51.) 80 n. •
Lewin, K. R. (1910.) "Nuclear relations of Paramecium caudatum during the

asexual period." {Proc. Cambridge Phil. Soc. Vol. xvi. p. 39.) 80.
Loisel, G. (1903.) " Experiences sur la conjugaison des Infusoires." {C. R. Soc.

5^;. T. LV. p. 53.) 130 n.
. (1903 a.) " Sur les causes de senescence chez les Protozoaires." {Ibid. \>. bb.)

130 n.
. (1903 6.) "Sur la senescence et sur la conjugaison des Protozoaires." {Zool.

Anz. Bd. XXVI. p. 484.) 130 n.
McClendon, J. F. (1909.) " Protozoan studies." {Journ. exp. Zool. Vol. VI. p. 265.)

58, 86.
Matheny, W. a. (1910.) "Effects of alcohol on the life-cycle of Paramecium."

{Ibid. Vol. VIII. p. 103.) 53 n.

188 On the Genetics of the Ciliate Protozoa

Madpas, E. (1888.) " Recherches experimentales sur la niultiiilication des

Infusoires cilies." {Arch. Zool. e.vp. T. vi. (2' sdr.) p. 165.) 16 — 22, 12.').
. (1889.) " Le rajeunissement karyogamique chez les Cilife." {Ibid. T. vii.

(2" s(5r.) p. 149.) 5 n., 6 u., 11, 15 n., 16—22, 91 n., 96, 121 nn., 125.
Metcalf, M. M. (1909.) " Opalina, etc." {Arch. /. J'rotistenL Bd. ■am. i>. Wb.)

9n.
MiDDLETON, A. R. (1913.) 'M\'ork on genetic ])roblems in Protozoa at Yale."

[Review of work by Woodrutt' and as.sociate.s, witli bibliogi-aphy.] {American

Nat. Vol. XLVii. p. 434.) 39 n.
Moody, J. E. (1912.) "Observations on the lifu-history of two rare ciliates,

Spathidium spathula and Actinobolus radian.^." {Juurn. Morph. \'ol. ,\xili.

p. 349.) 36, 50.
MuLSOW, W. (1913.) "Die Conjugation von Stentor coerulens luid Stentor poly-

murphus." {Arch. f. Prtitixtenk. Bd. xxviii. p. 363.) 7 n., 8, 91, 121.
Neresheimer, E. (1907.) "Die Fortpflanzung dor Opalinen." {Ibid. Suppl.

Bd. I. ]). 1.) 9 n.
. (1908.) "Der Zeugungskreis des Ichthyophthirius." {Ber. kgl. buyer, biol.

Versuchsstat. MUnchen, Bd. I. p. 165.) 9 n.
Pearl, R. (1907.) "A biometrical study of conjugation in Paramecium."

{Biometrika, Vol. v. p. 213.) 90, 112.
Peebles, F. (1912.) "Regeneration and regulation in Paramecium cavAatum."

{Biol. Bull. Vol. XXIII. p. 154.) 77, 88.
Peters, A. W. (1904.) " Metaboli.sni and division in Protozoa." {Proc. American

Acad. Arts and Sciences, Vol. xxxix. p. 441.) 52, 59.
POPOFF, M. (1907.) " Depres.sion der Protozoenzellc und der Geschlechtszellen der

Metazoen." {Arch. f. Protistenk: Suppl. Bd. I. p. 43.) 32, 95 n.
. (1908.) " E.xperinientelle Zellstudien." {Arch. f. Zellforsch. Bd. i. p. 245.)

45, 46, 55, 67.
. (1908a.) "Die Ganietenbildung luid die Conjugation von Carchesium

polypliiam L." {Zeitschr. W'iss. Zool. Bd. Lxxxix. p. 478.) 7, 95, 118 n.
. (1909.) " Experiraentelle Zellstudien. II. Ueber die Zellgrosse, ihre

Fixierung und Vererbung." {Arch. f. Zellforsch. Bd. in. p. 124.) 48, 66 n.,

67, 75, 76.
. (1909 a.) " Ueber den Einfluss chemischer Reagentien auf den Funktions-

zustand der Zelle." {S.B. Gesellsch. f. Morph. a. Physiol. Miinchen, Bd. xxv.

p. 55.) 34.
. (19096.) " E.\perimentelle Zellstudien. III. Ueber einige Ursachen der

physiologischen Depression der Zelle.'' {Arch. f. Zellforsch. Bd. iv. p. 1.) 34.
Powers, J. H. and Mitchell, C. (1910.) "A new species of Paramecium

{P. multimicronucleata) experimentally determined." {Biol. Bull. Vol. xix.

p. 324.) 78 u.
Prandtl, H. (1906.) "Die Konjugation von Didinium uasutum (). F. M." {Arch.

f. Protistenk. Bd. vii. p. 229.) 7, 13, 93, 95.
ProwazeK, S. (1904.) "Beitrag zur Kenntnis der Regeneration und Biologie der

Protozoen." {Ibid. Bd. iii. p. 44.) 76 n., 79, 88.
. (1904a.) "Degenerative Hyperregeueration bei den Protozoen." {Ibid.

p. 60.) 88.

C. DOBELL 189

Prowazek, S. (1909.) " Formdimorphismus bei ciliaten Infusorien." {Mem. Inst.

Oswaldo Cruz, Bd. i. p. 3.) 72.
Putter, A. (1905.) " Die Atmung der Protozoen." {Zeitschr. f. allg. Physiol.

Bd. V. p. 566.) 54.
Rautmann, H. (1909.) "Der Einfluss der Temperatur auf das (Jrosseiiverhaltnis

des Protoplasmakorper.s zuni Kern." {Arch. f. ZMforsch. Bd. III. p. 44.) 55, 56,

67, 68.

Simpson, J. Y. (1901.) "Observations on binary fi.ssion in the life-history of

Cihata." {Proc. Roy. Soc. Edinburgh, Vol. xxiii. p. 401.) 27, 82, 88, 122.
. (1902.) "The relation of binary fi.ssion to variation." [With a note by

Karl Pearson.] {Biometrika, Vol. I. p. 400.) 62.
Sun, a. (1912.) " Experimentelle Studien iiber Infusorien. (Vorl. Mitteil.) "

{Arch. f. Protistenk. Bd. xxvn. p. 207.) 35, 41, 55, 79.
Tatem, J. G. (1870.) "A contribution to the teratology of Infu.soria." {Monthly

microscop. Journ. (April), p. 194.) 81 n.
Wallengren, H. (1901.) "Zur Kenntnis des Neubildungs- und Rosorptions-

processes bei der Teilung der hypotrichen Infu.sorien." {Zool. Jahrb. (Anat.

Abt.), Bd. XV. p. 1.) 61.
. (1901a.) " Inanitionserscheinungen der Zelle. Untersuchungen an Pro-
tozoen." {Zeitschr. allg. Physiol. Bd. I. p. 67.) 94.
Waiters, F. A. (1912.) "Size relationships between conjugants and non-con-

jugants in Blepharisma uiidulans." {Biol. Bull. Vol. xxni. p. 195.) 90.
Woodruff, L. L. (1905.) "An experimental study on the life-hi.story of

hypotrichous Infusoria." {Journ. exp. Zool. Vol. ii. p. 585.) 37.
. (1908.) "Effects of alcohol on tlie life-cycle of Infusoria." {Biol. Bull.

Vol. XV. p. 85.) 53.
. (1908a.) "The life-cycle of Paramecium wlien subjected to a varied

environment." {American Nat. Vol. XLll. p. 520.) 39, 90 n.

. (1909.) In: Proc. American Soc. Zool. (jSci'tiHce, March 12, p. 425.) 39.

. (1909«.) " Further studies on the life-cycle of Paj-aniecjMHi." {Biol. Bull.

Vol. XVII. p. 287.) 17 n., 22 n., 39.
. (1911.) "The effect of culture medium contaminated with the excretion

products of Paramecium on its rate of reproduction." {Proc. Soc. e.vp. Biol.

Med. Vol. VIII. p. 100.) 41.
. (1911a.) "The effect of excretion products of Paramecium on its rate of

reproduction." {Journ. e.vp. Zool. Vol. x. p. 557.) 41.
. (1911?).) "■Paramecium aurelia and Paramecium caudatum." {Journ.

Morph. Vol. xxii. p. 223.) 39 n., 78.

. (1911c.) " Two thousand generations of /"araraeaMjn." {Arch. f. Protistenk.

Bd. XXI. p. 263.) 39.

. (1911 rf.) "Evidence on the adaptation of Paramecia to different environ-
ments." {Biol. Bull. Vol. XXII. p. 60.) 39, 43.

. (1912.) "A five-year pedigreed race of Paramecium -v/ithoxxt conjugation."

{Proc. Soc. exp. Biol. Med. Vol. IX. p. 121.) 39.

190 On the Genetics of the CUiate Protozoa

Woodruff, L. L. (li)12a.) "A sumniary of the results of certain physiological

studies on a pedigreed race of Paramecium." {Biockem. Bull. Vol. I. p. 396.)

39.
. (1913.) " Dreitausend und dreihundort (Jcnerationoii von I'tiramechim ohno

Konjugation oder klinstliche Reizung." (liial. Centralhl. x.x.xiil. I'd. p. 34.) 39.
. (1913(f.) "The efJ'ect of e.xcretion products of Infusoria on the same and

on different species, with special reference to the protozoan sequence in in-
fusions." {Journ. exp. Zool. Vol. xiv. p. 575.) 39, 41.
. (1913 6.) "Cell size, nuclear size and the nucleo-cytoplasmic relation

during the life of a pedigreed race of 0.x-ytricha fallax." {Ibid. Vol. xv. p. 1.)

38.
. (1914.) "On so-called conjugating and non-conjugating races of Paramecium."

{Ibid. Vol. XVI. 11. 237.) 39, 96, 101.
and Bait.sell, G. A. (1911.) "The reproduction of Paramecium aurelia in

a 'constant' culture medium of Vjeef extract.'' {Ibid. Vol. xi. p. 135.) 40.
. (1911 «.) " Rhythms in the reproductive activity of Infusoria." ylhid.

\<>\. XI. p. 339.) 40.
. (1911 6.) "The temperature coefficient of the rate of reproduction of

Paramecium aurelia." {American Journ. Phydol. Vol. xxix. p. 147.) 55.
and BuNZEL, H. H. (1909.) "The relative toxicity of various salts and acids

toward Paramecium." {Ibid. Vol. xxv. p. 190.) 54.
ZwEiBAUM, J. (1912.) "La conjugai.son et la differenciation sexuelle chez le.s

Infusoires (Enriques et Zweibaum). V. Les conditions neces.saires et suffisantes

pour la conjugaison du Paramecium caudatum " {Arch. f. Protistenk. Bd. xxvi.

p. 275.) 99.

A NOTE ON WHELDALE AND BASSETT'S PAPER
"ON A SUPPOSED SYNTHESIS OF ANTHO-
CYANIN'."

By ARTHUR ERNEST EVEREST, M.Sc, Ph.D.

Before the above-mentioned paper of Whelclale and Bassett came
to hand, the author had forwarded for publication elsewhere, a paper,
the contents of which very largely answer the criticism directed by
Wheldale and Bassett against his earlier paper (Roy. Soc. Proc. 1914, B,
vol. 87, p. 444). There are however a few points to which, in view of
Wheldale and Bassett's publication, the author feels it necessary to
draw attention.

It must be pointed out that as the result of the recent publication
of Willstatter and his collaborators (Sitzber. K. Akad. Wiss. Berlin,
1914, XII, 402) wherein the chemical examination of anthocyan pigments
(obtained pure and crystalline in every case, both as glucoside and non-
glucoside) from no fewer than ten flowers or fi'uits was briefly described,
and in which it was conclusively proved that these pigments were not

HO-

Cl

\/\^/

C-OH

OH

I
H

HO-

OH

\
C-H

C-OH

./

n

1 Journal of Genetics, 1914, Vol. iv. No. 1, p. 103.

192 Oil a Supposed Synthesis of Aiifhocyanin

oxidation products of flavonols, but were indeed related either to
/3-phenyl-benzo-7-pyrone or ;8-phenyl-benzo-a-pyrone, in the manner
suggested by the present author {loc. cit), and were derivatives of
either I or II, it becomes necessary that the work of Wheldalc and
Bassett upon the pigments of Antirrhinum should be continued until
crystalline substances having the characteristics of chemically pure
compounds are obtained, and these examined, before it can have any
weight in favour of the hypothesis which those authors support.

Doubtless Wheldale and Bassett were neither aware of the publica-
tion of Willstatter and his collaborators, nor present when Professor
Willstatter lectured on this subject at the University College, London,
in May last.

It is perhaps advisable to note that in the passage quoted by
Wheldale and Bas.sett, from the author's paper, viz. : " one would expect
that by taking the yellow gluco.'iide, hydrolising, then reducing \vith
removal of sugars, the anthocyanidin produced would combine with the
sugar present to form an anthocyanin. This is not the case," the

original reads: " , then reducing xvithout removal of sugars, "

{loc. cit. p. 445).

The authoi's recent work proves at least that Rutin — a disaccharide
of quercetin — passes by reduction, without hydrolysis at all, into a red
disaccharide pigment whose properties are those of an anthocyanin.
No satisfactory evidence is available to suggest that, in general, higher
glucosides of these pigments occur in plants, and until such evidence is
forthcoming, hypotheses demanding their existence are not likely to
receive much support from chemists.

It appears necessary to point out that the author does not use the
solubility or insolubility of these pigments in amyl alcohol as a test to
distinguish between anthocyanidins and anthocyanins, the test used
depends upon the distribution between dilute aqueous sulphuric acid
and amyl alcohol, coupled with the change in such distribution when
a glucoside pigment is hydrolysed ; this was clearly described in the
author's paper (loc. cit).

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The Problem of Individuality In OrganiiimB. l5y ('usuhtM Makkiso

The Development of u New Synttt-m of Or($anlc Chemistry,
Baaed on DUsodatlon Concepta. Uy .ions L'luic Nkf, with

t.),'; ", <,|,':/iUI<,;i '-f .1. VV. h, hl.Mlfl'.U).

The Living Oyoada. Uy C»a«i-k» .htnv.i'u Chamiikklaix.
iyi«rf;hiifi)<,«i of D«:l;iytd Germination In Beed*. I',y Wu.uam Cmdukvm.
The RlKldlty of the Earth and of Material*. iJy Alkkut A,

Ml'.lfKIA'/.'i'

The Problem of Fertilization, liy l•'('.A^K It Lim.ik.

Linear Integral Equallonx in General Analyals. I'-y Kuakim

llAHTIVfiN MoOUK.

The IJnIverHity <i\ Chicaffo Press

Ihe Camhridxe University Press

Aj(cntH for the IJriti«h limpirc

IvOndon, I'etter Lane

Volume IV

JANUARY, 1915

No. .s

A GENETIC AND CYTOLOGICAL STUDY OF
CERTAIN TYPES OF ALBINISM IN MAIZE.

New v,,,.,.^
«otan/c .1

By frank C. miles,

United States Department of Agncultun', Washinffton. D.C.

»/

Preinoxts Investigations.

It has loMi;- hoiMi noted thai while seedlings, •.\m\ variegiited unes ns
well, occasionally occur among many of our connnmi plants, hui only in
recent years has any systematic study of the problem been made.

Baur(l, -1) in UK)7 (later, in IDOS) reported liis study of the ytdlow-
leaved type of Antirrhinnni and Pelargoniinn. The yellow plants are
not capable of assimil.'itino' carbon dioxidi', .nnd eonscHpiently they can-
not live. This type of plant can exist only as a hybrid with a yreen
race, 'riie hybrid is an inli'nsi\(' yellow or " .\iire,-i-K;irbe," .and in the
second generation one-fourth of the plants are pure green which breed
true in succeeding generations, two-fourths are of the "Aurea" type
which segregate again in the next generation, and one-fourth are pure
yellow plants which theoretically should breed true, a test, however,
being impossible because the yellow plants always die in I lie seedling
stage.

In 1909 Correns(6) reported examples of what, has beei\ called the
"chlorina" type of plants in Mirahiiis jtthtpa. and in i'rtiea piliilijrra.
Haur(.'}) later repoi'ted the same type in Antirrliiiniiii. In these eases
the colour stutV (including the xant.hophyll and eai-ot-in ,-is well as the
chlorophyllin) seiMns tn be reduced in intensity, .•uid also ap|)e,Mrs to be
dinunished in quantity. In inheritance this "chlorina" character
behaved as a simple Mendelian recessive.

In llie same pa|)er ((i) Correns also reporteel a peculiar manner of
inheritance of leaf colour in Alirabilis Jala pa. He states that, the
leaves of the plants are irregularly spotted with yellowish white, a

.Tciuiii. of (li'u. IV 18

194 Albinism in Maize

condition which he terms " albomaculata." Occasionally there may
appear a plant which is either wholly gi-een or wholly white.

Anatomically it appears that this variegation is due to the fact that
in the whitish areas of the leaf the chromatophores are not green, but
are more or less bleached. The boundary between the green portion
and the white portion of a leaf is not sharp and distinct, but is gi-adual ;
the cells on the boundary may contain pale-green chromatophores, and
even in the same cell the intensity of colour of the different chromato-
phores may vaiy. A pure green branch remains so, and one which is
pure white remains white.

Seed from one of the gi-een branches produces green seedlings which
in further generations breed true. Seed from white branches produce
white seedlings which soon die because of their inability to perform
photosjoithetic processes. Seed from a variegated branch produces
some seedlings which are yellowish-white, some which are gi-een, and
some which are spotted or variegated.

In crosses Correns found that this variegated condition was inherited
only through the mother. When a flower on a white branch was
pollinated with pollen from a normal green plant, only white seedlings
were produced. And in the reciprocal cross, when a flower on a green
branch was pollinated with pollen from a flower of a white branch of
the variegated plant, only green seedlings were produced. But the
seedlings were known to be hybrids because of the behaviour of other
characters, e.g., colour of the flowers. These gi-een hybrids bred true
to the green character in succeeding generations, while the other
characters behaved as ordinary hybrids.

Baur(4) believes that in Primula sinensis he has a case analogous
to that described by Correns in Mirabilis, but at the time of his publica-
tion he had not fully investigated it.

Soon after the account of Correns concerning the case of non-
Mendelian inheritance in Mirabilis, Baur (-5) described another peculiar
form of non-Mendelian inheritance in Pelargonium zonule. Among
these plants there sometimes occur bud sports which are full}- white.
Seeds from the flowers on a white branch produce pure white seedlings.
Also the white-leaved form remains constant if grafted upon a green
plant.

Reciprocal crosses between this white-leaved form and a constant

green form gave seedlings which had a mosaic design of green and

white. Baur makes it apparent that in the ^i there was a vegetative

^segregation, the plants being composed of green and white mosaic areas.

F. C. Miles 195

This vegetative segregation concerned only the leaf colour. When a
white-leaved type and a green-leaved type were crossed and the parents
differed in some other character, e.g., flower colour, then the flower
colour Mendelized regularly.

In 1910 Baur (4) reported the occurrence of pure white-leaved plants
in Melandrium, Antii'rhinnm, and Phaseolus vulgaris. He had not fully
investigated the case of Phaseolus, but in both Melandrium and Antir-
rhinum the white condition proved to be a simple recessive character.
He believes that in Antirrhinum several factors are concerned in the
formation of leaf colour.

Analogous examples of the inheritance of the variegated condition
of leaves have been reported by Baur (4) for Aquilegia vulgaris, and by
CoiTens(6) for Mirahilis jalapa. The leaves of the plants have irregular
spots of a very light-green, or almost white, colour. Crossed with green
plants the F^ is green and F^ segregates into approximately three green
plants to one variegated plant. The variegated individuals when self-
pollinated usually breed true to the variegated condition, but occasion-
ally a fully-green plant occurs. Some of these green plants when self-
pollinated yield only green progenies, but others segregate into green
plants and variegated plants. The cause of this apparent inconstancy
of the variegated segregates is not fully understood.

East and Hayes (9) in 1911 mentioned the occurrence of striped
leaves in some of their cultures of maize. The striped plants were
apparently heterozygous, and when crossed with normal green plants
green was dominant. Progenies from striped plants consisted of green
plants, striped ones, and plants lacking chlorophyll. These latter died,
but, as the authors suggested, very probably were homozygous recessives.

Emerson (10) in 1912 reported his studies of various degrees of
chlorophyll reduction in maize, and described a number of types which
he had not fully investigated. He found pure white seedlings void of
chlorophyll, and which therefore died at any early age. In inheritance
he showed the character to be a simple Mendelian recessive.

In some of his cultures there were individuals which were almost
void of chlorophyll, but there was sufficient colouring material to give
the seedlings a yellowish-white appearance. Seedlings of this type
may turn more or less greenish, but at the time of his publication none
of the plants had lived longer than seven weeks. This character also
behaved as a simple Mendelian recessive.

The third type which he described concerned the partial reduction
of chlorophyll in growing and mature plants, resulting in a distinct

13—2

196 Albinism in Maize

yellowish-green colour of thf leaves. Crosses between these yellowish-
green individuals and fully-gncn plants showed that the character was
not a dominant one.

Another class included plants which had prominent longitudinal
stripes of green, pale green and white. So far as his study had gone
this character appeared to be a Mendelian recessive, though he men-
tioned that in certain cultures it appeared as if it might be due to the
absence of two positive factors.

There was also a peculiar, definite, regular striping of the leaf In
the region of the fibrovascular bundles the leaf is dark green, but
between the bundles the leaf is pale gi-een. He believed this was also
a Mendelian recessive.

He also reported the occurrence of plants the leaves of which
showed a few very narrow streaks of white. This type of inconspicuous
variegation was noted in a family in which pure white plants also
occurred. The character appeared to be a recessive one, but it had
not been fully studied.

Gernert(12) in 1912 briefly discussed the occurrence of pure white
seedlings in certain of his cultures of maize. He also reported the
presence of yellowish-gi'een plants and suggested the possibility that
they might be an intermediate stage of albinism. His studies clearly
indicate that the albino form is inherited as a simple Mendelian
recessive. The data in regard to the yellowish-gi-ecn condition were
rather incomplete, though it is quite likely that this is also a simple
recessive.

Davis (7) in 1912 described etiolated rosettes of Oenothera, which
he discovered in the second generation of a cross between two green
plants. The plants later became green and developed to maturity. In
a later paper (8) he reported that selfed seed from one of the F., jjlants
which had developed fi-om an etiolated seedling gave a progeny con-
sisting entirely of etiolated rosettes. The plants developing from these
etiolated rosettes became green, as did those of the preceding generation.

Nilsson-Ehle (13) in 1913 described the appearance and behaviour
of pure white and yellowish-white seedlings in some of the common
cereals.

In barley he found pure white plants and also some plants whose
leaves were yellowish, or sometimes slightly greenish. The individuals
of both types die in the seedling stage, though the yellowish ones lived
somewhat longer than those which were pure white. Green appeared
fully dominant, and in some families which consisted of homozygous

F. C. Miles 197

green plants and heterozygous green plants the two classes could not
be distinguished by any visible character. Progenies of heterozygous
plants consisted of three green plants to one white one.

In rye he noted the pure white-leaved type of plant, and also a
distinct yellow-leaved type, which in inheritance is analogous to the
white type. The plantlets of both types are incapable of surviving the
seedling stage. Green is dominant in both cases, and there is no "aurea"
type such as that described by Baur in Antij-rhinum, and also in Pelar-
gonium. He believes that the white condition of the leaves is due to
the omission or suppression of some factor, and that the yellowish con-
dition is due to the suppression or omission of a second factor.

He reported having found white plants among his cultures of oats,
but no record of the behaviour of the character was given.

The Inheritance of Albinism in Maize.

The experimental work which is hei-e reported was conducted at the
Nebraska Agricultural Experiment Station under the direction and
supervision of Dr R. A. Emerson, who had begun investigations on these
problems, and who kindly placed at the writer's disposal the records of
the work so far as it had progressed, together with the seed for con-
tinuing it. The writer takes this opportunity to express his appreciation
of the gi-acious assistance and many valuable suggestions which Doctor
Emerson has given him. The colour drawings reproduced here were
made by Miss Carrie M. Preston of the Nebraska 'Agricultural Experi-
ment Station, to whom the writer desires to express his thanks for this
assistance.

Since Emerson (10) has shown conclusively the recessive nature of
the pure white plants (PI. VIII, fig. 1), little attention has been given to
the inheritance of this case of albinism other than to count the white
and the green plants in each progeny where both types have occurred.
In the fourteen families that have been grown since the spring of 1912
there was a total of 361 green plants and 130 white ones, which approxi-
mates very closely the theoretical ratio of three green plants to one
white one.

More attention has been given to the case of the yellowish-white
individuals (PL VIII, figs. 3, 4 a, 4 h, 5 and 6). In the twelve pro-
genies which threw both green plants and yellowish-white ones there
were 726 green plants, 260 yellowish-white ones, and 5 which were
striped. It was possible to self-pollinate two of the striped plants, but

198 Albinism in Maize

only one of them set an ear. Seed from the self-pollinatetl ear gave
26 green plants and 16 which were yellowish- white. This is fully in
accord with the results obtained by Emerson, foi' the tests which he
made of similarly occurring striped plants led him to the conclusion
that the striped individuals were merely heterozygous green plants in
which the green was not fully dominant.

Emerson (10) mentioned that these yellowish-white plants some-
times become greenish, and one individual in the greenhouse lived to
the age of seven weeks. Perhaps the conditions out of doors were more
favourable for the development of these peculiar yellowish-white plants,
for during the season of 1912 several of them became fully green. The
position of some such jjlants was so well designated that there was no
mistake as to their identity, and two of the individuals were self-
pollinated. From the ears of the two plants 278 seedlings were grown
in the greenhouse, 277 of which were of the yellowish-white type, and
one was green. Undoubtedly the green plant was the result of an
accidental foreign pollination, for it is possible that a single pollen
grain from some green plant may have come in contact with one of
the silks. The fact that the yellowish-white plants breed true affords
additional evidence that a Mendelian recessive is being dealt with.

From the study of the pure white plants and the yellowish-white
ones it seems that both are simple recessives, yet it appears that more
than one factor must be concerned. Believing that there were different
factors involved, crosses were made between gi-een plants from a f;imily
which had thrown pure white seedlings, and green plants from a family
in which yellowish-white plants had occurred. It was hoped thus to
secure crosses of heterozygous green plants which, if self-pollinated,
would throw 25 per cent, pure white seedlings, with heterozygous green
individuals which, if self-pollinated, would yield 25 per cent, yellowish-
white seedlings in the next generation. The green plants which were
crossed were also self-pollinated. In Table I are indicated only those
crosses in which both parents were shown to be heterozygous.

It is interesting to note that the first generation of the cross between
the heterozygous green plants of the two categories consisted of 362
fully-green plants and one which was very faintly striped. From this
it appeal's that some factor which was lacking in one parent must have
been present in the other parent, in order that all the F^ plants should
be green. Otherwise it would be expected that in such a cross one-
foui'th of the F^ plants would not be fully green, owing to the fact that
the cross was between two heterozygous plants each of which, when

F. C. Miles 199

self-polliuated, yielded a progeny about one-fourth of which was non-
green.

TABLE I.

The result of self-pollinating and also of crossing tivo types of
heterozygous green platits.

idigree Number

Green

3008 (33)

28

3004 (10)

8

3008 ( 3)

32

3004 (18)

8

3008 ( 6)

48

3004 (18)

8

3008 (19)

36

3005 ( 3)

47

Striped

id parent plants

Fi progeny w
plants were

hen same
crossed

Yellowish-
white

Purfc white

Green

Striped

10

3\

106

V

10

6(

101

—

14

— 1
6(

58

—

— 10 — )

- - lel 97 -

As a working hypothesis it was assumed that the presence of two
factors is responsible for the processes which finally result in normal
green colour. For convenience, these factors will be designated by "A"
and " B," respectively. Then in the category in which pure white plants
occur let a heterozygous green individual be represented by the zygotic
formula AABh. When a plant of this nature forms its germ cells they
would be of two kinds, viz., AB and Ah. Upon self-pollination the next
generation would consist, on an average, of plants in the following ratio :
one green plant having the formula A ABB, and which in later genera-
tions would breed true green ; two gi-een plants having the formula
AABb, which in the next generation would segregate into three gi-een
plants to one white one ; and one white plant having the formula AAhh,
the white jjlant soon dying. Here, in the absence of the factor " B,"
the plant is pure white, the factor "A" being unable to bring about
the formation of any colouring material whatever.

In the category in which yellowish-white plants occur let the zygotic
formula AaBB represent a heterozygous green plant. If this plant be
self-pollinated, the next generation would consist of approximately three
green plants to one of the yellowish-white type. One-third of the green
plants would have the formula AABB and consequently would breed
true, while two-thirds of the green individuals would have the formula
AaBB and in the next generation would segregate in the regular
manner. The yellowish-white plant would be represented by aaBB.

' The striped individual had only a faint white stripe in one of its leaves. Otherwise
it was fully green.

200 Albinism in Maize

It seems that the factor " B" dififers from the factor "A" in that it
alone can bring about the changes necessary for the production of a
distinctly yellowish colour which later becomes slightly greenish, and
in some instances develo2)s into full green.

Continuing the hypothesis that the gametes arising from hetero-
zygous green plants of the category in which pure white plants occur
may be represented by AB and Ab, and the gametes arising from
heterozygous green plants of the category in which yellowish-white
plants occur may be represented by AB and aB, then by crossing the
two heterozygous green plants one would expect to obtain in Fi four
sorts of green plants in approximately equal numbers. The formulae
of the resulting F^ plants would be :

A ABB,

AABb,

AaBB,

AaBh.
By growing the second generation it can be shown experimentally
whether or not the first generation plants are of the four sorts enu-
merated in the foregoing. According to the hypothesis one of every
four Fi plants has the formula AABB, and in succeeding generations
should breed true gi-een. One of every four has the formula AABb,
and, this being identical with one parent, it should yield a progeny of
green plants and pure white ones in the ratio of three gi-een to one
white. One of every four has the formula AuBB, and as this one is
identical with the other parent the succeeding generation should consist
of gi-een plants and yellowish-white ones in the three-to-one ratio. One
plant of every four has the formula AaBb, and since this one is hetero-
zygous for both factors it would be expected to give a second generation
consisting of gxeen plants, yellowish-white plants, and pure white plants
in the dihybrid ratio of 9 : 3 : 4. In Table II are shown the hypothetical
formulae of the plants in the respective classes predicted in F^, together
with the expected behaviour in the third generation.

In order to make the experimental test, self-pollinated ears were
secured from eleven F^ plants which were gi'own in the gi-eenhouse.
The second generation results are shown in Table III.

It will be noted that the actual experimental results are very closely
in accord with the theoretical expectation fn)m the two-factor hypothesis.
That the F^ plants were of ft)ur distinct sorts, as regards the factors
concerned in formation of leaf colour, is shown by the respective F^

F. C. Miles

201

TABLE II.

Thu zygotic formulae and colour of plants in the progeny of F^ plants hetero-
zygous for both factors, together with the expected behaviour in F.^.

Fo plants

Zygotic
Number formulae

A ABB

AABb

AaBB

AaBb

AAbb

Aabb

aaBB

aaBb

aabb

Colour Expected behaviour in F,i

Green Should breed true

Greeu Should throw 3 greeu, 1 pure white

Green Should throw 3 green, 1 yellowish-white

Green Should throw 9 green, 3 yellowish-white, 4 pure white

P.ure white Fa plants would die iu seeding stage

Pure white F2 plants would die in seedling stage

Yellowish-white Survivors (by becoming green) should breed true

Yellowish-white Survivors (by becoming green) should breed true

Pure white F2 plants would die in seedling stage

TABLE III.

Results obtained in F„ of the cross between heterozygous green plamts of the
category in which pure luhite plants occur and heterozygous green plants of
the category iw ivhich yelloivish-white plants occur. Hypothetical formulae
of the respective F^ plants shoitm at tike left.

Hypotlietical formulae

of tlie Pedigree ,

respective Fi plants Number Green

AABB 3402 69

3405 6

Number of Plants

Yellowish-white

Striped 1

Totals

75

—

—

—

AABb

3401

65

—

18

2

iJ

8403

28

—

11

—

1»

3411

16

—

5

—

Totala

.. 109

—

34

2

AaBB

3406

68

26

—

2

>»

3407

31

8

—

—

„

3410

63

22

—

—

Totals

.. 162

56

—

2

AaBb

3400

25

5

10

—

»»

3408

52

17

23

—

..

3409

55

14

23

—

Totals

132

36

56

1 Previous tests have shown that similarly occurring striped plants were merely hetero-
zygous green plants, and no doubt the striped plants listed in this column should be
included with the green plants.

202 Albimsm in Maize

families. Two of these progenies' consisted only of green plants ; three
consisted of green plants and pure white ones in the ratio 3"2 : 1 ; three
were composed of green plants and j^ellowish-white ones in very nearly
the three-to-one ratio ; and three progenies yielded green plants, yellow-
ish-white ones, and pure white ones in a pro])()i-tion closely approximating
the 9:3:4 ratio. Theoretically the individuals of the four classes of F^
plants should occur in equal numbers. Of the eleven self-pollinated
Fy ears tested, three classes were represented by three each and one
class by two. This result is as near the theoretical expectation as
would be possible in a test of eleven ears.

Thus from the study of crosses in the foregoing description, between
plants of a family in which pure white seedlings occur and those of
a progeny where yellowish-white ones are found, it appears that the
production of normal green may be due to the presence of at least
two factors : in the absence of one the plant is pure white, while if
the other be absent the plant is yellowish-white, often becoming
greenish and occasionally, after a time, fully green.

Further tests have been made with the yellow-green plants (PI. VIII,
fig. 7) described by Emerson (10). In this category the seedlings have
the normal green colour at first, but later the leaves turn a distinct
yellowish colour and because of the striking resemblance to the "golden"
varieties of some of the horticultural plants the term " golden " will
hereafter be used in describing this type.

In the summer of 1912 the F.> of a cross between a green plant and
a golden one yielded 40 green jjlants and 11 golden ones. Two of the
green plants were crossed with pure golden plants and in one of these
crosses the green plant was heterozygous, as shown by the fact that the
progeny resulting from the cross consisted of 50 pei' cent, green plants
and 50 per cent, golden ones.

None of the F^ plants showed any of the golden colour, thus
indicating that green is completely dominant. Eight of the F^ plants
were self-pollinated. It would be expected that seed from each of the
Fi ears would yield a progeny consisting of 75 per cent, green plants
and 25 jDer cent, golden plants, but the progeny from one ear was
composed entirely of green plants. Many seedlings of this progeny
were destroyed by mice and only 12 plants grew to the age of eight
weeks, or the time when the golden individuals can usually be identified.
Since only one out of four plants should be of the golden type, it is

1 One of these progenie.s (No. 3iOo) consisted of too few individuals to show con-
clusively that it was of the pure green type, yet it is probable that such was the case.

F. C. Miles 203

barely possible that there might be 12 green plants without the
occurrence of any golden ones. But there is also the possibility that
the single grain which gave rise to the F^ plant was accidentally either
self-pollinated (the cross was green % x golden ^) or crossed by a
grain of pollen from a green plant. Seed from seven of the F^ ears
gave 230 green plants and 67 golden plants. This is a ratio of 3-4 : 1
instead of the expected 3 : 1 ratio, but since nearly all of the plants
were grown in the greenhouse under rather crowded conditions not all
of the golden individuals may have been identified.

A cross of an F^ plant with one of the golden plants yielded some
interesting results. The resulting ear had 195 grains which lacked
aleurone colour, and 36 grains which had black aleurone. When seed
lacking aleurone colour was planted there resulted 15 green plants and
15 golden ones, just as is expected when an F^ plant is crossed with the
recessive parent, but the seed having black aleurone gave eight plants,
all of which were green. It would be interesting to note whether or
not there is any coupling between the factors concerned in the develop-
ment of green in the leaves and the various factors involved in the
formation of aleurone colour.

Several of the golden plants were self-pollinated, but only three
small ears resulted as this type of plant rarely produces ears. The
three ears produced progenies of 4, 20, and 21 plants, respectively, and
all 45 plants were of the golden type. This, together with the various
other tests herein reported, indicates rather clearly that a simple
Mendelian recessive is being dealt with.

The cross between ordinary green plants and striped plants of the
race known as Zea japonica (PL VIII, figs. 8«, Hb) is affording interesting
study. The first generation is green, and when self-pollinated yields
a second generation consisting of both green plants and striped' plants
similar to the japonica parent. The results thus far obtained indicate
that the percentage of striped segregates in a progeny depends, to some
extent at least, upon whether or not aleurone colour is developed in
the grains planted. In the original cross part of the grains lacked
aleurone colour, and part of them had dark aleurone. These were
separated and F^ plants were grown from each lot.

In the second generation the family which developed no aleurone
colour in the grains consisted of 94 green plants and 30 striped ones,

' At first the striped segregates, as well as the individuals of Zea japonica, cannot
be distinguished from green plants, but about five to eight weeks after planting the new
leaves begin to show the prominent longitudinal stripes.

204 Albinism in Maize

approximating closely the monohybrid ratio. Fifteen of the green
plants and five striped ones were self-pollinated. Ordinarily one would
expect the striped segregates to breed true, and of the green plants
one-third should breed true green, while two-thirds should segregate
into green plants and striped plants in the ratio three green : one
striped. Of the fifteen progenies grown from self-pollinated ears of
the green F.^ plants five consisted only of green plants ; nine consisted
of both green plants and striped plants, the total numbers being
262 green and 86 striped ; and one progeny consisted wholly of striped
plants. The fact that one progeny consisted of striped plants can
easily be accounted for, because in the original cross one of the parents
was a very dark-purple plant and in the F.^ there were a number of
purple plants appearing. It was noted during the summer of 1912
that it was often difficult to distinguish the striping on some of the
purple plants, and in occasional cases it might have been overlooked.
The records show that the individual which gave a progeny of striped
plants was a dark-purple plant, and consequently it may have been
faintly striped, but the fact was overlooked when notes on plant colour
were taken. Of the five progenies grown from the five self-pollinated
striped segregates four bred true to the striped condition, there being
288 plants in all four families. But one progeny consisted of 34 striped
plants and eight green ones. This condition appears somewhat similar
to the results obtained by Baur(4) and Correns(6) in crosses between
variegated and green plants, for they reported that not all of the
variegated segregates bred true. Emerson(ll) reports results of a
comparable nature in the case of variegation in pericarp colour of maize
grains. As regards plant colour, there is the possibility that the green
individuals were the result of accidental cross-pollination of the varie-
gated plants with green plants.

The second-generation family descending from those grains which
in the original cross had dark aleurone consisted of 87 green plants and
10 striped ones. Thirteen gi-een plants and five striped ones were
self-pollinated. The results from these pollinations are shown in
Table IV.

It is seen that too large a percentage of the green plants bred true
green for this to be interpreted as a simple case of inheritance. Also,
only one of the striped plants bred true to the striped condition, and
the fact that four of the striped plants threw some green individuals
indicates that the green plants probably were not due to accidental
cross-pollination. The peculiar behaviour in this case is not understood

F. C. Miles

205

at present, but careful study should be made to determine whether
or not some of the factors for aleurone colour may in some way be
related to the factor (or factors) concerned in the development of
the variegated leaves, for in the instances in which aleurone colour
was not involved the variegated condition of leaves appeared to be a
simple Mendelian recessive.

TABLE IV.

Colour of F„ plants and the aleurone colour of grains home on the self-pollinated
F.-. plants, together with results obtained in F^ of the cross betvOeen Zen
japonica and ordinary green maize.

Results obtained in F:i

Colour of
Fz plants

Green

Colour of aleurone in
grains of ear of F^ plants

Lacked aleurone colour

Number of
green plants

Number of
striped plants

30

—

36

—

39

—

39

—

26

11

51

12

25

7

21

6

Striped

3 black grains

to
1 non-coloured one
9 black grains

to
7 non-coloured ones
Black aleurone colour

Lacked aleurone colour

9 black grains

to
7 non-coloured ones

27

30
45

57
35
31

13

5

16

12

27

31'

6
16

63
57
32
24
23

A study has been made of the behaviour of the peculiar green-
striped plants (PI. VIII, fig. 9) described by Emerson (10). When
crossed with green plants the Fj plants are fully green, and in the F^
there occurs the regular segregation. In a total of 466 plants of
nine progenies there were 357 green plants and 109 which had

1 The parent which yielded this progeny of striped plants was also one of the purplish
plants, and if any stripes were present they were so very faint that they were overlooked
when notes were taken.

20f> AlMnisw in Maize

the characteristic green striping. This approximates very closely the
3 : 1 ratio and indicates that a simple Mendelian recessive is being
dealt with.

Crosses between plants of the different categories were made as
follows :

Ze(( jciponica x golden.

Green-striped x golden.

Green-striped x yellowish-white, later turning green.

In tliese cases neither parent in any of the crosses was fully green,
yet all the F-^ plants resulting from each cross were of normal green
colour. The F., generations were grown from the respective crosses,
and notes were taken concerning the seedlings until five weeks after
date of planting. Since the japonica-striped plants, the green-striped
ones, and the golden ones cannot be identified with extreme accuracy
until they are from eight to ten weeks old, it was unfortunate that no
notes could be taken at a later date. But the absence of the writer
from the State during the remainder of the growing season prevented
such notes being taken.

At the date when the last notes were made, however, one could
distinguish individuals of the various categories. In the F.. of the
cross between plants of Zea japonica and those of the golden type
there were 102 green plants, five of the japonica type, 37 golden ones,
and four golden plants striped after the manner of Zea japonica. The
second generation of the cross between green-striped plants and golden
ones consisted of 63 green plants, 14 green-striped ones, 22 of the
golden type, and four golden plants which had the peculiar striped
pattern of the green-striped category. In F„ of the cross between
green-striped and yellowish-white plants there were 146 green plants,
25 green-striped, and 42 yellowish-white. In these cases there were
too few individuals in the various F., families for one to place much
dependence in the ratios, yet it seems probable that they arc merely
modifications of the dihybrid ratio. A further study of these crosses
must be made, in order to fully understand the relation between the
various types.

An Anatomical Study of the Leaves of Certain Types of Maize.

At the suggestion of Professor Emerson the writer undertook a
histological study of the leaves of some of the various types of albescent
maize. It was soon found that a more satisfactory study could be made

F. C. Miles 207

if the specimens were first killed and then prepared according to the
paraffin method.

Experiments were made with several different killing reagents, but
the most satisfactory method was to allow the leaf sections to remain
for about two and one-half hours in a modification of Carnoy's killing
fluid composed of absolute alcohol 50 per cent., chloroform 25 per cent.,
and glacial acetic acid 25 per cent. After killing, the specimens were
washed in absolute alcohol, cleared in xylol, infiltrated and embedded
in paraffin.

Cross sections of the leaves were made of from three to fifteen
microns in thickness, but those sections which were either ten or twelve
microns in thickness proved the best for a study of the plastids. Of the
stains employed Lichtgriln appeared to be most satisfactory, yet some
good results were obtained from Delafield's haematoxylin, and also from
acid fuchsin. Camera lucida drawings were made from the prepared
slides by Miss Lucille Goodloe, Washington, D.C., to whom the writer
desires to express his thanks at this time.

Examination of leaves from the pure white plants showed that the
plastids apparently were almost, if not entirely, lacking (Fig. L). Even
leucoplasts could not be differentiated with any of the stains which
were used. This is an extreme condition, yet the conclusion that no

Fig. 1. Cross section through a leaf of a
pure white seedling, such as is illustrated
in PI. VIII, fig. 1. No plastids could be
differentiated, x 360.

plastids are present is substantiated by the fact that these pure white
plants have never been known to turn green, or even greenish, in colour,
but always die as soon as the young seedlings have exhausted the food
stored in the kernel planted.

The condition found, however, in the leaves of the yellowish-white

208 Albinism in Maize

plants is somewhat different. Preparations were made of leaves of
different ages. Fig. 2 shows the cross. section of a leaf of a very

Fig. 2. Cross section through a leaf of a .young yellowish-white
seedling at the stage illustrated in PI. VIII, fig. 3. The
plastids are small and are comparatively few iu number.
They were differentiated in the same manner as the plastids
of the guard cells of the stoma shown at " S." x 380.

young seedling such as that shown in PI. VIII, fig. 3. Even though the
leaf was only yellowish, there was a differentiation of small granular
bodies resembling diminutive plastids. It will be noted that these
small bodies react to the stain in the same manner as do the plastids
shown in the guard cells of the stoma. The material illustrated in
Fig. 3 was taken from an older plant which was turning greenish.

Fig. 3. Cross section through a leaf of an older yellowish-
white plant, such as illustrated in PI. VIII. fig. 4a. The
plastids have increased in size and number, x 360.

F. C. M1LE8

209

Hero it is seen thut the granular bodies in the cells have increased
both in size and in niunber. In some cases, perhaps under especially
favourable conditions, the plants became almost, if not fully, green
(see PI. VIII, fig. (i). Preparations from the leaves at this time appear
as shown in Fig. 4. Here apparently normal plastids are present, and
they resemble very closely those found in ordinary green corn leaves
(Fig. 5). Thus in the case of the yellowish-white plants which may

Fig. 4. Cross section through a leaf ot a still older yellowish-wbite
plant which is becoming greenish. Leaf from which the
section was taken was almost green, x -llO.

Fig. .5. Cross section through a leaf of
a normal green plant, x 380.

Jouru. of Oen. iv

14

210 Albinism in Maize

turn greenish or green the studies indicate that the gradual develop-
ment from small and comparatively few granular bodies into apjwrently
normal plastids is synchronous with the changing of the plants from
the yellowish-white condition of the young seedlings to the green
condition of the older plants.

This condition differs from that in plants which are yellowish-white
because of being grown in darkness. Seed which was known to produce
only normal green plants was j^lanted in a flat and was germinated in
the dark. The young seedlings were yellowish in colour (no green
showing), and preparations of the leaves showed that plastids were in
abundance (Fig. 6).

Fig. 6. Cross section through a leaf of a
seedling made yellowish by being germi-
nated and grown in darkness, x 380.

The leaves of the race of maize known as Zea jrqyonica have stripes
of dark green, pale green, and white ( PI. VIII, fig. 86). In the dark-green
area of these leaves chloroplasts are present in approximately the same
number, and are about the same size as in ordinary green leaves (Fig. 7),
while in the white portion there are very few, if any, plastids. There
were noted, however, a very few small bodies which may have been
leucoplasts. The pale-green stripe afforded an interesting study.
Fig. 8 shows the position of the plastids which are present. They
are seen to be in only those cells next to the lower epidermis. Since
this is the case, the intensity of the green colour is diminished as the

F. C. Miles 211

light passes through the several layers which are void of plastids.
It was noted that the stripe which appeared a pale green on the
upper surface of the leaf was of a more intense green colour on the

Fig. 7. Cross section through a greeu portion of a leaf
of the striped race Zea japonica. Plastids are dis-
tributed through the different cell layers. x 3.50.

Fig. 8. Cross section through a pale green stripe of a
leaf of the striped race Zea japonica. Apparently
normal plastids are found in the cells near the
lower epidermis, and light passing through the
colourless layers above causes a reduction of
the intensity of the green colour, x 300.

lower surface. This would be expected because if the leaf be viewed
from the lower surface the cells containing chloroplasts would be
immediately underneath the epidermis, and consequently the green
would show more vividly than if the light had ti) pass through several
layers of cells.

212 Albinism in Maize

Fig. 9 shows a portiou of a white stripe and also a portion of a
green stripe in a variegated leaf of maize. It is not at all difficult to
note the boundary between the white and the green portion.s, for well-
differentiated plastids are present in the green portion and are ab.sent
in the white portion.

Fig. 9. Cross section through a striped green and
white leaf. The boundary between the green and
the white stripes is distinct, as is shown by the
absence of plastids in the white portion, x 380.

Summary.

From the studies of the various categories it appears that in all
cases, with the possible e.xception of the striped leaves in Zea japonica,
the several degrees of albinism in corn leaves behave as simple
Mendelian recessives; the first generation of a cross with ordinary
green races giving fully green plants, and the second generation
segi'egating in the ratio of three green plants to one plant of the
particular type which was used in the cross.

The study of the manner of inheritance of variegated leaves of
Zea japonica in crosses where aleurone colour is involved has not
been completed.

A rather definite relation has been pointed out between a pure
white type -of maize plant and a yellowish-white type, the results
indicating that the presence of at least two factors is necessary for
the development of normal green in the leaves of maize. In the

F. O. Miles 213

absence of one of these factors the phmt is pure white and soon dies,
while in the absence of the other factor the plant at first is yellowish-
white, but is capable of developing into a greenish condition and
sometimes into a pure gi-een plant.

Studies of the relation between the other categories have not been
completed. Crosses of striped plants of the japonica type with golden
plants, and those of the green-striped plants with golden plants, and
also the crosses of green-striped plants with yellowish-white individuals
which turn green have all resulted in first generation plants which were
of the normal gi-een colour. Although it was impossible to note the
second generation plants, except during the first five weeks of their
growth, it was possible at that time to identify segregates of the
respective categories. The results secured in these crosses, however,
add further evidence to the hypothesis that more than one factor is
concerned in the production of normal green cohjur in the leaves of
maize. Apjjarently there is lacking in each parent some genetic
factor (or factors perhaps) which is concerned in the development
of chlorophyll, and, since the F^ plants are normal green, it appears
as if that facti.>r which is lacking in one parent may be present in
the other.

In the pure white plants no plastids could be differentiated. In
the yellowish-white plants which later may become green plastids
apparently are present from the fii'st, although they are few in number
and are very small, gradually increasing in number and size as the leaf
turns green.

In Zea japonica the manner of distribution of plastids may be
compared with the condition which Trelease (14) has described in
certain variegated Agaves. He found that the normal green condition
was due to the presence of plastids in the subepidermal region of the
leaf. In variegated leaves, if the stripe was pale greenish, there was
found to be a suppression of jalastids through several of the subepi-
dermal cells, while in a pure white stripe there was " all but complete
suppression of recognizable plastids."

EXPLANATION OF PLATE.

PLATE VIII.
Fig. 1. Pure white seedling.
Fig. 2. Ordinary green seedling.

Fig. 3. Yellowish-white seedling 12 days after planting.
Figs. 4« and ib. Yellowish-white seedlings 15 days after planting.

214 Albinism in Maize

Fig. 5. Yellowish-wlutc scedliiii,' wliicli is tuining greenish; 20 days after plautinf,'.
Fig. 6. Yellowisli-wliite seedling wliicli has become nearly t;reen ; 2".) days after planting;.
Fig. 7. Golden plant. As the plants rrrow older they become even more golden in

colour.
Fig. Ha. Striped plant of Xea japonica.
Fig.Hh. Section of a leaf of Zea japonica.

Note the stripes of white, yellowish, pale green and dark green. Fi^s. 7 and 8

in the text show the distribution of plastiJs in the dark-;jreen and in the

pale-green stripes, "r/" and "p," respectively.
Fig. 9. Section of a leaf of a green-striped plant.

LITERATURE CITED.

1. Badr, E. " Unter.siichungen iiber die Erblichkeitsverhiiltnisse einer luir in

Bastardform lebensfiihigen Sippe \'ou Antiriidnuin nmjus." Ber. d. IJeutsch.
Bot. Ges. Bd. XXV. liJOT.

2. . "Die Aure;i-Sippen von Antirrldnuia niajus." Zeitschr. f. ind. Ahs. it.

Venrbungslehre, Bd. I. 1908.

3. . " Vererbungs- und Bastai'dieruug.sver.suche mit Antirrhimtm iivtjus."

Zeitschr. f. ind. Ahs. u. Vererlmngslchre, Bd. III. 1910.

4. . " Untersuchungen iiber die Vererbung von Chromatophorenmerkmalen

bei Melandriuin, Antirrhinum uud Aquilegia.' Zeitschr. f. ind. Ahs. n.
Vererhungslehre, Bd. iv. 1910.

5. . " Das Wesen und die Erblichkeitsverhaltnisse der ' varietates albo-

ina,rgma,ta,Bhort' von Pelargonium zonale.'" Zeitschr. f. ind. Ahs. u. Verer-
hungslehre, Bd. I. 1909.

6. C0RREN13, C. " Vererbungsversuche mit blass(gelb)griinen und bunlblattrigen

Sippen boi Mirabilis Jalapa, Urtica pilulifera und Lunaria annua."
Zeitschr./. ind. Ahs. v. Vererhungslehre, Bd. I. 1909.

7. Davis, B. M. " Genetical Studies on Oenothera." III. /I men iVai. July, 1912.

8. . "Genetical Studies on Oenothera." IV. .4mer. Nat. August and

September, 1913.

9. East, E. M. and Hayes, H. K. " Inheritance in Maize." Conn. Agr. E.cpl.

Station, Bull, clxvii. 1911.

10. Emerson, R. A. "The Inheritance of Certain Forms of Chlorophyll Reduction

in Corn Leaves." Nuhr. Agr. Expt. Station, Twenty-fifth Animal Report,
1912.

11. . " The Inheritance of a Recurring Somatic Variation in Variegated Ears

of Maize." Amer. Nat. Vol. XLVlir. pp. 87 — 115, 1914; Ncbr. Agr.
Expt. Station. Research Bull. iv. pp. 1 — 3.5, 1914.

12. (JiERNERT, W. B. The Auali/sis of Characters in Corn and their Behaviour in

Transmission. Published by the author, 1912.

13. Nii.sson-Ehlb, M. "Einige Beobachtungen iiber erbliche Variationen der

Chlorophylleigcnschaft bei den Getreidearten." Zeitschr. f. ind. Ahs. u.
Vererhungslehre, Vol. ix. 1913.

14. Thki.ease, William. ^^Variegation in the Agaveae," Sonderabdruck aus der

" Wiesuer- Festschrift," 1908. (Verlag Carl Konegen in Thien.)

JOURNAL OF GENETICS, VOL. IV. NO. 3

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IP'

\-

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Al>

\

5

PLATE VIII

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\

i^

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'J

STUDIES IN THE PHYSIOLOGY OF
FERTILIZATION.

PART I. ON THE CONDITIONS OF SELF-FERTILIZATION

IN GIONA.

By H. M. FUCHS

(Shuttleworth Student of Gonville and Cwius College, Gamhridge).

CONTENTS.

PAGE

I. Introduction ......... . • 215

II. Methods; importance of exactness ..... . 221

III. Factors influencing the extent of self-fertilization ..... 232

1. Sperm-concentration ......... 232

2. Length of time that eggs and sperm lie in water before fertilization 234

3. Position of genital products in their ducts ..... 237

IV. The effect of a sperm suspension on the eggs of the same individual . . 240
v. Comparison of the subsequent development of eggs self- and cross-fertilized

under various conditions ......... 244

1. Eggs cross-fertilized at different intervals after the removal of the

genital products from the body of the animal .... 244

2. Eggs and sperm from different parts of genital ducts . . . 249

3. Comparison of development after self- and cross-fertilization . 249
VI. Summary of experimental results ........ 253

VII. Conclusion 254

Beferences ............. 258

I. Introduction.

The main problems presented by the fact of self-sterility — that is
to say, the difficulty or impossibility of fertilizing the female gametes
of certain hermaphrodite animals and plants with male gametes derived
from the same individuals — can be divided under five heads. First, the
determination of the degree of self-sterility shown by the individuals

liK) Studies in the rinjdohMpi of FertiUzatioii

of a given race ; second, what factors influence the degree of self-
sterility ; third, the causes of the self-sterility; fourth, the inherit-
ance of self-sterility ; and fifth, the effect of self-fertilization on the
offspring. The present investigation was commenced in the belief
that IJiamt intestitiuliH is almost, if not coniplet(>ly, self-sterile, in which
case the first, seci.md, and fifth of the problems just eiuinierated would
be non-existent. It was proposed to investigate the inheritance of
the self-sterility on the lines of the extremely interesting work b}^
Correns (4) on Cardamine pratensis. That is to say, separate cross-
fertilized families would be raised to maturity, and then the membei-s of
a family be crossed inter se, with individuals of the family derived from
the reciprocal cross and with unrelated individuals.

Castle (1) originally demonstrated the self-sterility of Ciona in-
testinalis when making fertilizations to obtain stages in the development
of the embryo. The animals used were from Newport. A number of
specimens were isolated in separate dishes, where they discharged eggs
and sperm simultaneously nearly every day. In the majority of cases
none of these eggs were found to have been fertilized, although in some
as many as 90 "/„ segmented. In contrast with this, when several
animals were placed together in an aquarium, usually 100°/^ of eggs
which were discharged segmented. Repeating the observations with
eggs and sperm artificially removed from the genital ducts, he found
that self-fertilization usually caused few or no eggs to segment, although
in one instance 50 " j ^ were self-fertilized. Similar observations were
made by Gutherz in 1903 (7a).

During the course of an extensive investigation on the phenomenon
of self-sterility in Ciona, Morgan never found such high percentages of
eggs self-fertilized as Castle had done. The first part of Morgan's work
was done at Woods Hole in 1903, when he states (9, p. 138) with
regard to artificial fertilizations, that " I have rarely seen more than
from one to ten per cent, of self- fertilized eggs segment, and in the greater
number of cases not a single egg segmented.'' The second part of the
work was done at Coronado Beach, California, in 1905. Here, in makingr
fertilizations w"ith eggs and sperui removed artificially from the bodies
of the animals, " in only one case out of many hundreds of eggs mixed
with their own sperm did fertilization occur." The final part of the
investigation was carried out at Woods Hole in 1909 (11), and this
time cases of self-fertilization were exti-emely rare.

Castle himself suggested that when high self-fertilization per-
centages were obtained, tho result may really have bc^en due to the

H. M. Fuoiis 217

C7'oss-fertiliKation of soiiie <jf the eggs by .spermatozoa accidentally
present in the water. Morgan (9) thinks this probable, since he never
got such high percentages as Castle. Nevertheless, in two cases Castle
obtained 90 7o of eggs self- fertilized after the animals had been isolated
for three days, and it is very unlikely that spermatozoa of Ciona can
retain their power of fertilizing after having been for such an extended
period in sea-water, even if they are, as Morgan suggests, entangiefl in
the branchial basket of the animal.

Apart from experiments to be described below, in which fertilizations
were made with spermatozoa that had been in sea-water for varying
intervals, and all of which showed a more or less rapid deterioration of
the sperm, the following preliminaiy tests showed me that the activity
of the spermatozoa does not last for very long after they have left the
vas deferens.

(1) Unfertilized eggs were removed from four animals at 7 p.m.
At the same time, sperm was removed from the sperm-ducts of the
same individuals and a mixed suspension made up. At 11.20 a.m.
on the following morning, some of each of the lots of eggs were
fertilized with sperm ju.st removed from another individual, and in
all four cases 100 /,^ of the eggs segmented. Other samples of these
eggs, however, when inseminated at the same time with the sperm-
suspension made on the preceding evening, showed only one irregularly
segmenting egg between them. Evidently the life of unfertilized eggs
is much longer than that of spermatozoa in sea-water. (2) Eggs were
removed fi-om an individual into sea-vyater at 8 p.m. Fertilized on
the following evening at 9.50 p.m., with fresh sperm, 100 "j ^ segmented,
although irregularly. (3) A comparatively dilute suspension was made
of the sperm of one individual at 8 p.m. At 9.50 p.m. on the following-
evening, this failed to fertilize a single egg of a sample just removed
from another animal.

It appears, therefore, that the higher self-fertilization percentages
obtained by Castle than by Morgan were probably due to other causes
than the presence of spermatozoa of other individuals, since the animals
had been isolated for some days.

Preliminary experiments (in which rigid precautions were taken as
described in the following section to exclude the presence of " foreign "
spermatozoa) soon convinced me that Ciona intestinalis at Naples was
far from being completely self-sterile, and that the degree of self-sterility
varied greatly. In many cases no eggs segmented after insemina-
tion with sperm of the same individual, in others comparatively high

218 Shidies in the Physioloijij of Fertilization

percentages of fertilized eggs were obtained. Potts (13, p. 481) has
already observed that the Ciona at Naples is not completely self-sterile.
He obtained "nearly 100°/^ of embryos" from self-fertilized eggs — an
occurrence which was comparatively rare in my experiments, but
which would be accounted for if Potts used very concentrated sperm
suspensions. Evidently the race of Ciona at Woods Hole behaves
differently in respect to its capacity for self-fertilization from that at
Naples'.

The first preliminary experiments were made on the lines adopted
by Castle. Animals were isolated for a number of days in bowls of
filtered water and the proportion of self-fertilized eggs in each lot
was counted. Although this was a fairly extensive series lasting over
a month, subsequent experience showed that it was valueless except
in demonstrating that self-fertilization can occur. The different lots
of eggs deposited by a given animal on successive days showed very
varying amounts of self-fertilization, a circumstance which undoubtedly
depended largely on the different amounts of sperm ejected with the
eggs. Further, the varying self-fertilization percentages suggested, but
by no means proved, that the eggs of one individual vary from day to
day in their capability of being self-fertilized. Subsequent experiments
proved this surmise to be true. (See pp. 236 et seq.)

The fact that Ciona at Naples is far from being quite self-sterile
and that the extent of the self-sterility varies in different individuals
and in the same individual with successive lots of eggs and sperm
produced, appeared at first sight to render the investigation of the
inheritance of the self-sterility difhcult or impossible. Nevertheless
self-fertilization proved always to be very much more difficult to bring
about than cross-fertilization. In general a comparatively dilute sperm
suspension, but one which was concentrated enough to fertilize 100 %
of eggs of another individual, would only rarely fertilize even one to
two per cent, of the eggs derived from the same animal as itself To
bring about self-fertilization much more concentrated sperm suspensions
were necessary, and even then it by no means always followed. Taking
this fact into consideration, it is plain that after the limits and con-
ditions of the occurrence of self-fertilization have been determined, the
problem of the inheritance of the comparative self-sterility is still a
feasible one for investigation.

' Whether tlie form of Ciona iiitestiimlis at Woods Hole is really a distinct variety
(var. teiielln) or not seems to be uncertain, especially as the distinsuishiug characters of
var. feni-lla are not very definite (8, 12).

H. M. FucHS 219

Experiincnts are now in progress which are designed to give an
answer to the question of inheritance ; but the object of the work
described in Part I of this paper was to investigate thoroughly the
conditions under which self-fertilization occurs'. Besides this, the present
work deals with the effects of self-fertilization on the offspring. The
cause or reason of self-sterility is quite another problem and one which
is hardly touched upon here, although Section IV deals with a phe-
nomenon which may shed considerable light on the question.

Starting with a race of Gioiia which was almost completely self-
sterile, the main object of Morgan's work was to investigate the cause
of this phenomenon and to bring about self-fertilization by artificial
means. Besides this he carried out an extensive series of cross-
fertilizations to discover whether eggs of one individual can be fertilized
equally well by the sperm of all other individuals, provided that it is
in good condition. He concluded that this does not take place. A
critique of Morgan's methods is given in the following section, showing
that the way in which the experiments were carried out does not justify
the conclusions drawn from them. It is not denied that there may be
different degrees of sterility exhibited in cross-fertilizations, but Morgan's
experiments do not at all prove the point. The only undoubted result
which Morgan seems to have established with regard to the self-sterility
is that self-fertilization can be induced to a certain degree by ti-eatment
of the sperm (and possibly also of the eggs) with solutions of ether and
alcohol. It is difficult to see, however, what light this sheds on the
cause of the .self-sterility.

The preliminary experiments of the jjresent investigation brought
to light some interesting facts with regard to the manner of egg and
sperm deposition. The isolated animals lay some hundred of eggs
almost every day for a week or so, after which the frequency of the
depositions and the number of eggs laid decreases. Sperm is almost
always deposited simultaneously with the eggs, only rarely the one
being shed without the other. The manner in which the genital
products are ejected is as follows. A small mass of eggs exudes from
the aperture of the oviduct into the atrium, and at the same time a
small mass of sperm issues from the vas deferens. After about half

' In connection with the phenomenon of self-sterility in Ciona, the following record of
a hermaphrodite Echinoid is of interest. In March 1913 a specimen of SlrongyloceiUrotus
lividus was kindly given to me by Dr Meyerhof, in which three of the gonads were ovaries
and in the remaining two the upper third was testis and the lower two-thirds ovary.
Both eggs and sperm were ripe and the specimen proved to be perfectly self-fertile.

•220 Sfwh'es in tlw PlojHkohHpi of Fertilization

a miiiutu, when this mass of eggs and sperm lying at the apertures of
the genital ducts has reached a certain size, it is suddenly expelled
into the water through the exhalent siphon by a violent contraction
of the muscles in the body wall. The process is then repeated. The
whole period of egg and spei-m deposititm usually lasts fVom five to ten
minutes.

Castle (1) first observed that Ciona intestinalis deposits its genital
products at a definite time of day. His animals always discharged
their eggs and sperm an hour or an hour and a half before sunrise.
Morgan (9) states that at Woods Hole " the eggs are laid in the early
morning, at dawn." Seeliger (14), however, remarks that at Trieste
there is no such regularity in the time of deposition. With the Ciona
at Naples, 131 depositions of genital products were observed by me
in a number of isolated animals during the months of December and
January. 105 of these took place in the late afternoon after 5 o'clock,
that is to say after dusk had set in. Six depositions were in the night
(as judged by the state of segmentation of the eggs on the following
morning), and 20 in the early morning, about sunrise. Almost all the
early morning depositions were by individuals which had been laying
regidarly for a number of days in the late afternoons, which suggested
that morning laying was an effect of the laboratory conditions. As
this was not an invariable rule, it is hardly of value to publish the
exact i-ecords. The only conclusion to be drawn is that in the great
majority of cases eggs and sperm were discharged in the late afternoons,
and that in no cases did the depositions occur during the period of
bright daylight. It is hoped to decide whether the stimulus to dis-
charge the genital products may be given by a change from light to
darkness, or from darkness to light, by further laboratory experiments,
which will also deal with the mechanism of ejection.

The present investigation was carried out at Naples between
December 1912 and July 1913, while I was occupying the Cambridge
University Table. I would like to take the opportunity of tendering
my very best thanks to the Staff of the Zoological Station, both for
the valuable advice and help afforded me throughout the course of the
work, and for the trouble taken to procure an abundant supply of
material, which was brought in fresh from the sea almost every day.
My thanks are also due to Mr R. H. Comf)ton of Uonville and Caius
College, Cambridge, who originally suggested to me ;ui investigation
of the problem of self-sterility.

H. M. FucHS 221

II. Method.s. Ijiportance of Exactness.

Before detailing the technique employed, the essential nature of
the method of experimentation should be explained. Shortly put, it
was as follows. Approximately equal numbers of eggs from a single
animal were placed in two or more dishes, each of which contained
exactly the same quantity of sea-water. Fertilization was then effected
by the addition to each dish of exactly the same amount of sperm-
suspension from a single animal. The object was to discover the effect
on the eggs of different treatments of those in the different dishes, or
the effects on the spermatozoa of varj'ing preliminary treatments of the
different .samples to be added to the dishes. If this preliminary handling
consisted in adding a certain solution to the quantity of sperm-sus-
pension to be used for fertilizing the eggs in one of the dishes, the
consequent dilution of the suspension was compensated for by the
addition to the other lots of sperm of equal quantities of plain sea-
water. The criterion of the effects on the spermatozoa of this preliminary
handling consisted in a comparison of the jjercentages of eggs fertilized
when the differently treated but equal quantities of sperm-suspension
were added to the dishes containing the eggs.

From this outline a number of absolutely necessary precautions can
be deduced :

(1) If the experiment consists in the comparison of the effects of
a different treatment either of the eggs or of the spermatozoa, the eggs
in the different dishes must all be derived from one individual, or no
legitimate comparison can be made. Similarly, the sperm used to effect
the fertilizations must all be derived from one animal, and moreover
the definite quantities used must all be drawn from the same sus-
pension. The reason for this is that it is found to be impossible in
practice to make up two sperm-suspensions of equal concentrations.
The only method of judging the concentration of .spermatozoa in a
suspension is by the degree of milkiness, as seen with the eye. Differ-
ences in the degree of milkine.ss which are undetectable by the eye
may mean considerable variation in the concentration of the sperma-
tozoa (as measured by the percentages of eggs fertilized by equal
quantities of the different suspensions).

(2) An experiment can be made to compare the proportions of
eggs fertilized when equal numbers are taken from different indivi-
duals, and to each lot is added an exactly equal amount of one sperm-

22'2 Studies in the PhyHiolo^m of Fertilization

suspension. The reciprocal experiment of fertilizing equal quantities
of eggs from one animal with equal amounts of sperm-suspensions from
several other individuals does not give a valid result. This again is
owing to the fact that it is impossible to make up different sperm-
suspensions of exactly equal concentrations.

(3) The way in which the spermatozoa are subjected to the dif-
ferent treatments is as follows. A suspension is made up and afterwards
divided among two or more dishes, which can then be subjected to
different conditions of temperature, etc., or be kept for different lengths
of time before being used. If the treatment consists in the addition of
some substance, equal amounts of the one suspension are tried out into
several dishes, and to each dish is added an equal volume of liquid con-
taining the substances. One dish is kept as a control of untreated
spermatozoa, and to this is added a like volume of plain sea-water. In
this way the concentrations of spermatozoa are kept identical in the
various subdivisions of the suspension.

(4) In making the fertilizations in the different dishes of an
experiment, it is essential that all the eggs in each lot should have
an equal chance of being fertilized by the spermatozoa which are added.
This is brought about by pouring the water containing the eggs and
spermatozoa from one dish to another immediately after the sperm has
been added. This pouring was usually done ten times backwards and
forwards for each lot of eggs, which ensures a thorough mixing of the
water in which the eggs were lying with the sperm-suspension. The
definite quantities of the latter were measured out with a graduated
pipette, or by a certain number of drops from a pipette of uniform bore
closed at the top by the thumb.

(5) Since the experiments consisted in comparing the percentages
of eggs fertilized under different conditions, it is obvious that these
percentages must be above 0 and below 100. If none or if all of the
eggs are fertilized in each of the dishes, no comparisons can be made.
In consequence, the concentration of the sperm-suspension employed
must be such that some but not all of the eggs are fertilized. Now
this is a difficult matter to arrange, since the amount of dilution
necessary in making up a sperm-suspension so that some only of the
eggs will be fertilized when a definite (]uantity of the suspension is
added to them must be judged by the eye. In practice this can be
done with more or less success, but very often the experiment turns
out a failurt'. More than twice as many experiments were made as
are recorded below. All those not detailed were failures because the

H. M. FucHS 223

sperm -suspension was made either so weak that 0 "/^^ or less than 1 °/^
(< 1 °/^), or so concentrated that 100 /_ of the eggs were fertilized in
each of the dishes, thus allowing of no comparisons.

This applies to fertilization in the Echinoids and to cross-fertilization '
in Gioiia. In almost every case, provided that none of the eggs are
immature or pathological, 100 /^ fertilizations can be obtained if a
sperm-suspension of sufficient concentration is used. With regard to
self-fertilization in Ciona the matter is simpler, for it is very seldom
that 100 7o of the eggs can be fertilized by sperm from the same
individual, however concentrated the suspension be. On the other
hand, a comparatively large number of cases occur in which no self-
fertilization at all can be obtained.

The next important point preliminary to an investigation of this
nature is a determination of the degree of accuracy of the results. For
this purpose an extended series of trials was made, lasting over a month.
The object of these trial experiments was to discover the chief sources
of error and the ways of overcoming them, together with the extent to
which the numerical results were accurate.

It was found that accuracy depends on four main factors, which are
as follows :

(a) The mode of making up the sperm-suspension.

This was found to be the largest source of error. If a suspension
be made by taking some of the sperm from the vas deferens, mixing it
with sea-water, and then stirring the liquid, the spermatozoa are by no
means evenly distributed through the water. In the case of a thick
suspension this is easily seen with the naked eye, but with the dilute
suspensions used in the cross-fertilization experiments it is not apparent.
If equal quantities of liquid are taken from such a suspension and added
to equal amounts of eggs, the percentages of the latter which are fer-
tilized are usually by no means the same : the error may even be as
high as 20 °/,^. A number of ways were tried of getting a more even
distribution of the spermatozoa in the water, such as continued stirring,
shaking the suspension in a tube, pouring once or twice from one dish
to another, filtering — but all with no certain success. The results could
not be depended upon. Eventually, however, it was found that by pouring
the liquid to and fro ten times at least, a very uniform suspension could
be obtained, so that equal amounts fertilized equal proportions of eggs.

' Throughout this paper "cross-fertilization " means the fertilization of Ascidian eggs
by spermatozoa of another individual.

'224 Sfiidiefi in the Plniniohxpi of FeHilization

(b) Thoroiigli niLiiiKj of the eggs before dividing tlteiii, into differetd
lots.

The ni'cessity for this precaution will be obvious in the light of
exjieriments to be described below, which show that eggs taken from
different parts of the oviduct of an individual Cionn have very different
capacities of being fertilized.

(c) The importance of the way in xvhicli iiiseininatiuii is effected
has already been dwelt upon. A rapid and even distribution of the
spermatozoa through the wa,ter containing the eggs is essential, so that
all the eggs in the different dishes may have an equal chance of being
fertilized. The most accurate method was found to be as follows. A
certain amount of the sperm-suspension to be used was pipetted into
each of a number of dishes containing equal amounts (10 — 20 cc.) of
sea- water. The spermatozoa were thoroughly mi.xed with the water in
each of these dishes by repeated jjouring, after which the contents of
the dishes were poured on to equal amounts of eggs lying in a drop
or two of water in other vessels. The operation was finished by re-
pouring each of the lots a number of times. This was the method
adopted in most of the experiments, although sometimes the definite
amounts of sperm-suspension were simply pipetted straight into dishes
in which the eggs were lying in equal quantities of water. After this
the liquids were, of course, poured backwards and forwards several times
to ensure thorough mixing.

(d) The cownting of the percentages of eggs fertilized.

An investigation of which the results depend on a comparison of
the ratio between two classes under different conditions involves, of
course, an accurate estimation of this ratio. In our case it is not
sufficient to give a rough approximation of the proportion of fertilized
to unfertilized eggs as estimated by the eye. The whole investigation
is an exact one, and accuracy is as necessaiy in observing the results as
in carrying out the technique of the experiments.

The percentage of fertilized eggs in a given sample was ;Uways
calculated from counts made when the eggs were in the 4-cell stage.
This stage is reached at a convenient time after fertilization, and it is
impossible to mistake a segmenting for an unsegmented egg when the
former has divided into four cells. A very necessary preliminar}^ to
the operation of couriting is a thorough mixing of the eggs in each
dish. It is frequently found that eggs lying in one part of a dish show
a slightly different proportion of fertilized to unfertilized from those in

H. M. FucHS 225

another part. The actual counting was done by taking a number of
eggs up in a pipette and spreading them in a line along a glass slide,
which was then passed under a low power of the mici'oscope. Every
•percentage 7-ecorded is calculated from a count q/'400 — 500 egys. Since
most of the fertilizations were made with less than 2000 eggs, the
percentages obtained by counting 400 — 500 of these should be fairly
accurate. Each percentage is reduced to the nearest whole number.

The final result of these trials, in which each experiment was made
double or triple in order to test the degree of accuracy attainable, was
that the extent of the experimental error in the percentages might be
as much as 3 7, • It will be seen, however, that in the results of the
experiments, the differences between percentages to be compared with
one another were almost always considerably greater than the extent
of this error. There is one way, of course, in which an absolute control
can be kept of any experiment. Each experiment might be duplicated —
that is to say, each fertilization be made twice with the same quantities
of sperm and eggs — and the results rejected if they did not agree
exactly. This method was not adopted because time would not permit
of it in all cases, nevertheless a similar control was kept in many of
the experiments. Each of the fertilizations was made double, but
different quantities of sperm were added in the two cases. By both
giving similar differences in the percentages, the weaker-sperm and
stronger-sperm series in an experiment confirmed one another. This
method had the further advantage that there was much more prob-
ability of the experiment being a success. If in the weaker-sperm
series the sperm was so dilute that no eggs were fertilized at all,
probably the addition of a greater quantity to form the stronger-sperm
series would give percentages lying between 0 and 100, and permit of
comparison. Similarly, if it happened that the stronger-sperm gave all
100 7,, fertilizations, the addition of less sperm-suspension to the water
with the egg.s to form the weaker-sperm series might give a result
lying between 0 and 100 °/,^. If, however, both series lay between 0
and 100 °/^, they were a check on one another.

The above outline gives the way in which the experiments were
carried out, and the precautions adopted to ensure the maximum of
accuracy in the results. There is another and more obvious source
of error which has to be guarded against in all experiments on fer-
tilization. Each experiment is made with the eggs from a single
individual and the spermatozoa from a single individual. The presence

Jouiu. of Gen. iv 15

226 StncUes in the Physioloou of Fertilization

of even a minute quantity of spermatozoa of any other individual com-
pletely vitiates the results. This applies to cross-fertilizations as much
as to experiments on self-fertilization, but in the latter the effects of
such contamination are more obvious. It is with comparative difficulty
that self-fertilization can be brought about at all in Ciona, and when it
is found that this difficulty is lessened by the changing of a certain
factor, the elementary precaution of ensuring the CDinplete absence of
spermatozoa from any other individual must naturally be taken before
certainty can be attained that the alteration is really due tu the con-
dition which has been changed in the e.xperiment.

The precautions which were taken to ensure the absence of sperm-
contamination can be divided under three heads :

(1) Sterilization of instruments and luater.

All glass-ware was cleaned with hot water before being used.
Similarly scissors, forceps, etc. were dipped into hot water after every
operation. The sea-water used in the experiments was taken from the
circulation in the laboratory, and passed through a Berkefeld filter. The
complete absence of spermatozoa from such water was shown by the fact
that Ascidian or Echinoid eggs never segmented wh(,'n left in it without
the addition of sperm.

(2) Method of removing the eggs and sperm from the animals.
The first source of contamination of the genital products of Ciona

with the spermatozoa of other individuals is the possible jiresence in
the pharynx, branchial basket and atrium of the animals of spermatozoa
taken in with the water. To guard against this, the animals (which
were in almost every case brought in from the sea on the day they
were used) were treated as follows. The atrial cavity of each was slit
up by inserting one point of a pair of scissors into the exhalent aperture.
The animal was then pinned down and wa.shed with a copious stream
of fresh-water.

The second pos.sible source of contamination is that in removing the
eggs from the oviduct some sperm might accidentally be taken with
them from the adjacent vas deferens. It is extremely easy to puncture
the oviduct and then suck out the contained eggs with a pipette with-
out injuring the adjacent vas deferens at all. Sperm, however, will
always exude from the terminal aperture of the vas deferens during
this operation. In order to prevent this, the tops of the oviduct and
vas deferens were always tightl}' closed by means of a "bull-dog" clip
before the animal was washed under the stream of fresh-watei'.

H. M. FucHS -I'll

After the eggs had been taken from the oviduct, the latter was slit
open from end to end and washed out with sea-water to remove all
remaining eggs. The vas deferens was then punctured, and a quantity
of sperm removed.

(3) Controls of unfertilized eggs.

The final check on the possible presence of " foreign " spermatozoa
is kept by the unfertilized egg controls, which are a sine qua non to all
experiments on fertilization. Out of every lot of eggs used in the ex-
periments described below, 1000 or more were kept in a separate dish
to which no sperm was added. In no case recorded did a single egg
segment in the control.

In describing the experiments, the genital products of each animal
used are denoted by a single letter, the capital type (e.g. A) referring to
the eggs of a female and the small type (e.g. b) to the spermatozoa of a
male. The cross made by fertilizing the eggs A of one individual with
the sperm b of another is then written Ajb. In the case of a herma-
phrodite like Ciona, A eggs self-fertilized is written Aja, and A eggs
cross-fertilized with b sperm from another individual, Ajb.

The results of the experiments are tabulated, and in the Tables
the numbers given always mean percentages of eggs which have been
fertilized, unless otherwise stated.

In order to illustrate the most usual method of carrying out the
experiments an example is given here, which has the further advantage
that it shows up a possible source of error which might easily lead to
incorrect conclusions if not taken into account. If sea-water is passed
through a filter-paper, the first water to come through is distinctly acid.
Whereas normal sea-water gave yellow with neutral red, this filtrate
gave a pink colour. It will be shown later that small traces of acid are
very injurious to spermatozoa, so that if such filtered water be used in
the experiments the results will be quite untrustworthy. If a stream
of sea-water is run through a filter-paper for five minutes, after this
time the filtrate no longer shows any difference in acidity from normal
sea-water when tested with the indicator. The point of the experiments
to be described was to discover whether such filtered water still has
any effect on spermatozoa, due to the possible presence of some other
substance derived from the filter-paper.

The first experiment was made as follows. Eggs A were removed
fi'om one individual Ciomi and sperm h from another, by the method
already described. The eggs were placed in sterilized sea-water and

15—2

228 Studies in the Phyaiologij of Fertilization

thoroughly mixed by pouring from one dish to another. A sperm-
suspension was made and diluted down to the required amount, after
which it was thoroughly mixed by pouring it 20 times from one
dish to another. Two small glass dishes were then taken. Into the
first (1) was placed 10 cc. of normal sea- water, and into the second (2)
10 cc. of sea- water which had come through the filter-paper. This
paper had already been washed for 15 minutes in running sea-water
and the water which came through after this interval gave the same
reaction as normal sea-water with neutral red. To each of the dishes (1)
and (2) were added three drops of the sperm-suspension b, so that two
sperm-suspensions were obtained, (1) in normal sea-water, and (2) in
filter-paper water, and each of identical concentration. The liquid in
each dish was thoroughly mixed by pouring. Into each of two other
small dishes were then placed approximately equal quantities of A eggs
in three drops of water. The liquid in dish (1) was then poured on to
one of these lots of eggs, and that in (2) on to the other. By this means
fertilization was effected, and in order to ensure a quick and thorough
mixing of the eggs and sperm, the contents of the two dishes were each
poured backwards and forwards into two other dishes several times.
When the 4-cell stage was attained, the percentages of eggs which had
been fertilized in the two dishes were counted in the way described
above.

The experiment was then repeated on exactly similar lines with
another cross, Ajc. The results are tabulated below.

TABLE. To show the effect on the "fertilizing poiver " of a sperm-suspension
o/ water which has passed through a washed filter-paper. (44.26.6.)

(1) Normal (2) Filter-paper

water water

Exp. 1, cross A/b 100 77

Exp. 2, cross Ajr 95 31

The Table shows that in each of the experiments fewer eggs were
fertilized by the sperm-suspension treated with water from the filter-
paper than by the sperm not so treated, although the two suspensions
were of identical concentrations. In the first case the decrease was
from 100% to 77%, and in the second from 95 7,, to 31%. This is
expressed by saying that the "fertilizing power" of the sperm-suspension
had been diminished by the treatment with filter-paper water.

The foregoing remarks have emphasized enough the importance on
such work as this, where the validity of the results depends on numerical

H. M. FucHS 229

comparisons, of making the experiments under such conditions that
comparisons are strictly possible. In previous work, the results of which
have depended on the percentages of eggs fertilized under different
conditions, the method of experimentation has in general been for from
exact. This is often the case in Echinoderm hybridization work — and
numerous other examples might be cited, but the investigation which
particularly interests us here is that of T. H. Morgan on fertilization in
Ciona (9, 10, 11).

A considerable part of the work of this investigator was concerned
with the question as to whether the eggs of all individuals are capable
of being fertilized by the sperm of all other individuals. This is a
problem of peculiar interest, especially in view of recent work on self-
sterility in plants. It is far from being an easy point to settle, since
the difficulty of making up the sperm -suspensions of the different
individuals to be compared of even approximately equal concentrations
is great. That this equal strength of the different sperm-suspensions
is an absolutely necessary datum for interpreting the results is shown
by the fact that in making cross-fertilizations even small differences in
sperm-concentration produce large variations in the percentages of eggs
fertilized. Morgan's method was to select five or six individuals, make
all the possible reciprocal fertilizations between them, and then compare
the percentages of eggs fertilized in the different crosses. It is stated
(9, p. 148) that "The sperm, a, of the first individual was then taken
out and put into a small amount of water. It was then distributed to
one set of eggs from each of the other individuals, B, C, D, E; then the
sperm of B was taken out and applied to another set of eggs." It is
not stated, however, whether sperm b was of nearly the same concentra-
tion as sperm a ; nor is there any mention as to whether the eggs and
sperm were thoroughly mixed before fertilization, and with one another
at the moment of insemination. The observation is made, however
(10, p. 321), that in all cases enough sperm was probably used to fertilize
all the eggs capable of uniting with that sperm. In the first series (9)
several of the combinations were made twice in the same experiment,
but frequently gave very divergent results. Thus in Exp XIII, Ea
(i.e. E eggs with a sperm) gave first 100 °/„ and then 85 7o of fertilized
eggs. In Ex. XIV, Ea gave 0% and 4>°l^: Ae 0% and 100%. In
Exp. XV, Ea gave 2 7„ and 0 7„ : De 90 7, and 100 7„ : Ae 0 % and
100 7„. In Exp. XVI, Ea 5°/, and 70 7„: De 100 7„ and 30 7„: Ae
75 7o ^■nd 100 7o- It seems extremely probable that these divergencies
are due to inexact experimentation, although Morgan suggests (10,

230 Studies iti tlie Phjidoloffy of Fertilization

p. ;^20) that the extreme difference of 0 "/r '"''f' ")f*"/ in one combina-
tion in Exp. XIV anrl again in Exp. XV "is due no doubt to the faihu'e
to add sperm to the first lot, which might easily occur." How art' we
to judge of the exactness of the results of the crosses which were not
made twice over? The conclusion clrawn from 240 combinations was
that the sperm of an individual ('iaitd is not capable of fertilizing the
eggs of all other individuals to an equal extent, although some of the
results, according to Morgan, may have been due to the presence with
the eggs of "body-fluids." It would seem, however, that the equality
of conditions for each of the cro.sses should be made very much mon'
exact before this conclusion is justified.

The way in which the percentages of segmenting eggs were counted
is not given. Nor are there usually any details as to about how many
eggs were used in the experiments, nor how many of these were counted.
In some few cases, however, the number of eggs present is given in
brackets after the percentages. Apparently these were the cases in
which exceptionally few eggs were used, as the following citations would
seem to show. From the 1904 paper: p. 14.5, 0°/,^ (only 3 eggs);
p. 149, 0 7, (1 egg); p. 150, 10% (only 10 eggs); p. 151, 25% (only
4 eggs), 50 7, (only 8 eggs), 12 7„ (only 8 eggs), etc. From the 1910
paper: p. 218, 100 7„ (3 eggs), 0 7„ (1 egg); p. 219, 100 7„ (2 eggs),
50 7o (2 eggs), 100 7o (4 eggs)'. How many eggs were present in the
other lots is not stated.

In all work on fertilization under ditferent experimental conditions,
the complete absence of genital products of other individuals than those
used in a particular experiment is an absolutely essential condition.
This is, if possible, more than ever important in work on self-fertilization
in comparatively self-sterile organisms, where the presence of the most
minute trace of " foreign " spermatozoa upsets the whole results. The
importance of this precaution is fully realized by Morgan. He says
(9, p. 137), "The individuals to be used were isolated, as a rule, from
24 to 48 hours, and in most cases were rinsed in fi-esh-water before
opening," and that instruments were properly sterilized. Nevertheless,
throughout the work the expression of doubt recurs that such and such
a result may have been due to contamination with " foreign " sperma-
tozoa. There is no necessity for such doubt at all, for if unfertilized
controls are kept of every lot of eggs employed, and unfertilized eggs
are left lying in samples of every fluid used, if none of these segment

' On the same page is recorded " 60% (no eggs)," hut this must be a printer's error.

H. M. FucHS 231

there can be no reasonable question of the accidental presence of
spermatozoa which would vitiate the results.

Experiments were made by Morgan to test the influence of ovary-
extract on the extent of self-fertilizations. In two out of eight cases
some of the eggs segmented, but " there may have been contamination
in the latter case" (9, p. 139). Again, it was tried whether water in
which eggs had been violently shaken would favour the self-fertilization
of the eggs of another individual. In several cases self-fertilization did
occur in the presence of such water (10, p. 325); but "There is, in fact,
a source of contamination in this experiment that may fully account for
all the cases observed. In removing the eggs from the oviduct .some of
the sperm from the vas deferens may be accidentally squeezed out and
become mixed with the eggs, and remaining in the follicle water
fertilize the other eggs." Whj^ were not unfertilized eggs from another
individual left lying in the water in which the eggs had been shaken
(follicle water) to test whether it contained siJermatozoa or not ?

Again, the question recurs (11, p. 207) as to whether the few cases
of self-fertilization may not have been due to contamination with
" foreign " spermatozoa retained in the branchial basket of the animal,
which might then come into contact with the eggs removed from the
oviduct. But if unfertilized samples of these eggs had been kept as
controls the possible presence of such " foreign " spermatozoa would
have been made known.

The repetition of such examples serves no further purpose, but in
conclusion it should be pointed out that some of the experiments made
to yield results by comparison were not comparative at all. For
example, eggs were fragmented by pressui-e under a cover-slip and
many of the eggs which had been broken in this way were self-fertilized
on the addition of " own " sperm, thus " indicating that the resistance
to self-fertilization is due to something in the membranes surrounding
the eggs " (10, p. 326). But the second half of the experiment, which
would justify such a conclusion, is missing. It is not mentioned whether
some of the same lot of eggs, which had not been crushed, showed no
self-fertilization when inseminated with an equal amount of the same
sperm-suspension. Again, a number of experiments were made to see
whether subjection of the genital products to low temperatures would
increase the percentages of eggs self- fertilized. In some cases a
considerable proportion of eggs showed self-fertilization after such
treatment, but apparently the second half of the experiment was again
missing, in which some of the same lot of eggs untreated with cold

232 Studies in the Phystobxjii of Fertilization

should have bprn fertilized with a like ainount nf the same suspension
of "own" sperm, also untreated. Morgan concluded that the variation
in the extent of self-fertilization in the dificient experiments of this
series was probably due to contamination with sperm contained in the
branchial baskets of the animals from which the eggs and sperm had
been removed. But this possibility could have been tested by leaving
" foreign " unfertilized eggs in sea-water containing a piece of the
branchial basket.

III. Factors influencing the Extent of Self-fertilization.

From the foregoing it is plain that Ciona intestinalis at Naples is
not absolutely self-sterile, but that self-fertilization occurs to a con-
siderable degree. Moreover the extent to which it takes place is very
variable. The experiments described in this section are attempts to
analyse the factors which influence the extent of the self-fertilization ;
that is to say, they are exact comparisons between the proportion of
eggs to a given individual self-fertilized under a certain set of condi-
tions, and the proportion when one of the conditions is altered. At the
same time a comparison is made between the percentage of some of
the same lot of eggs cross-fertilized by sperm of a "foreign'" individual,
or of eggs of a " foreign " individual cross-fertilized by sperm of the first
one, under similar conditions to those used in the self-fertilizations.

1. Sperm-concentration.

Each of the first series of experiments made to test the influence of
sperm-concentration on self-fertilization was carried out as follows.

Appi-oximately equal amounts of eggs to a given individual were
placed in equal quantities of water in four dishes. Fertilization was
effected by the addition of sperm as follows : to dish (1), five drops of a
milky suspension of " own " sperm ; to dish (2), 4 cc. of ditto ; to (3),
five drops of a milky suspension of " foreign " sperm of approximately

TABLE I. (7.8.1.)

Eggs selfed Eggs crossed

Cross
Ajm
Bjm
Cjm
Djm

■ " Own " sperm is used to dsnotc sperm taken from the same individual as the
eggs: "foreign " sperm is that from anoilier individual.

Cross

5 drops
sperai

4cc.
sperm

Exp. 1. Aja

0

oH

Exp. 2. Bjb

0

22

Exp. 3. CIc

12

100

Exp. 4. Did

0

r,tj

5 drops
sperm

100

■tec.
sperm

100

100

100

100

100

100

100

H. M. FucHS

233

the same concentration as the suspension of " own " sperm ; to (4), 4 cc.
of ditto. The results of these experiments are shown in Tabic I.

The Table shows that in each of the e.xperiments an increase in the
concentration of " own " sperm caused an increase in the number of
eggs self-fertilized, the percentage in Exp. 3 being raised to 100, a
proportion which subsequent experiments proved to be uncommon.
The fertilizations of the eggs with " foreign " sperm demonstrate the
ease with which cross-fertilization is effected as compared with self-
fertilization by an approximately equal concentration of sperm, 100 °/^
of the eggs segmenting in each experiment, even with the more dilute
sperm-suspensions.

Further experiments were then carried out confirming this con-
clusion. The results are shown in Tables II, III and IV. The method
used in the experiments of Table II, which was the same as that used
in those of Tables III and IV, was as follows.

Six dishes were prepared, containing equal amounts of water. To
the first three were added approximately equal amounts of eggs E to be
selfed, and to the last three, approximately equal' amounts of eggs of
another individual F to be crossed. Fertilization was effected by the
addition to each dish of the amounts of sperm-suspension e given in
the Table. In this case the comparison between the proportion of eggs
selfed and the proportion crossed with the same amount of sperm was
exact, since the same sperm-suspension was used in each fertilization.

Cross

Eje

TABLE II. (8.9.1.)

Self-fertilization

2 drops
sperm

2

25 drops
sperm

24

10 cc.
sperm

100

Cross-fertilization

Cross
Fje

2 drops
sperm

100

25 drops
sperm

100

10 cc.
sperm

100

Cross
Gl9

1 drop
sperm

TABLE in. (8.9.1.

Self-fertilization

5 drops
sperm

20 drops
sperm

100 drops
sperm

11

ajg

1 drop
sperm

100

Cross-fertilization

5 drops
sperm

100

20 drops
sperm

100

100 drops
sperm

100

TABLE IV. (lO.U.l.

Self-fertilization

Cross

Cross-fertilization

L'ross

3 drops
sperm
+ 10 cc.
water

1 cc. 5 cc-
sperm sperm
+10 cc. +10 cc.

water water

10 cc.
sperm

3 drops

.sperm

+ 10 cc.

water

Ice.
sperm
+ 10 cc.

water

5 cc.
sperm
+ 10 cc.
water

10 cc.
sperm

Jh

<1

-=1

47

89

Kij

19

77

100

100

sin

0

0

U

43

Ojii

100

100

100

100

234 Studies in the Pkysiology of Fertilization

2. Length of time that eggs and sperm lie in water before fertiliz- .
ation.

The next factor investigated was the length of time the eggs and
sperm lay in water before they were brought together. The eggs and
sperm of one individual and the eggs of another were removed separately
from the genital ducts and fertilizations were made at definite intervals
after these genital products had been brought into the sea-water.

The exact procedure in Exp. 1 was as follows :

The genital products were removed at 2.55 — 3.10 p.m. : at 3.45 —
3.50' the first fertilizations were made. A certain quantity of eggs A
was placed in water, after which 3 cc. of a thick .sperm-suspension a
was added. At the same time an approximately equal quantity of eggs
D was placed in 10 cc. water in another dish and 1 cc. of a dilute sperm-
suspension a was added. In the previous section it was shown that a
considerably greater concentration of sperm is necessary to effect self-
fertilization than would cross-fertilize 100 % of foreign eggs, and
therefore the sperm a used for cross-fertilization with eggs D had to
be made much more dilute than that used to self-fertilize eggs A. The
details just given were repeated exactly at each subsequent fertilization,
the times of which are given in the Table below. The same sperm-
suspensions were of course used to effect the self- and cross-fertilization
after each interval.

The other four experiments were carried out on the same lines as
Exp. 1. The details of the quantities of sperm and water used and the
intervals at which the fertilizations were made are given in Table V.

As in all the experiments dealing with self-fertilization, more trials
were made than the successful ones I'ecorded below, owing to the fact
that there are always a certain number of individuals in which none of
the eggs will self-fertilize. In this series, five successful experiments
are recorded, but besides these there were three in which the self-
fertilization percentages were nil throughout.

The first point brought out by the experiments of Table V is that
in every case the percentages of eggs self-fertilized rose with succeeding
fertilization.s. In four cases it fell again after having reached a maximum,
but in Exp. 3 at 4^ hours after removing the eggs and sperm from the
animal the maximum had not yet been reached. The corresponding
cross-fertilizations with the sperm from the same animals showed in no
case a rise in percentages with successive fertilizations. In Exps. 2

' The fertilization times given in Table V were taken at the end of eacli operation,
which occupied 3 — 5 minutes.

H. M. FiTCHS 235

and 3 there was an excess of sperm throvighout in the cross-fertiliza-
tions, 100% of eggs segmenting every time. In Exps. 1, 4 and 5,
however, the percentages fell — in the last two, rapidly.

TABLE V.

Fert. Fert. Fert. Fert.

3.50 4.50 5.45 6.50

Cut outl 2.55-3.10 p.m. (19-5"C.) (20"C.) (20"C.) (20-5'C.)

Exp. 1 Self-fert. Aja, 3 cc. thick sperm, 10 cc. water 4 6 9 0

Cross-fert. Z)/a, 1 cc. dilute sperm, 10 cc. water 100 100 79 89-

Exp. 2. Self-fert. Clc 3 cc. thick sperm, 10 cc. water 21 23 — 12

Cross-fert. F/c, Ice. dilute sperm: 10 cc. water 100 100 — 100

Fert. Fert. Fert. Fert. Fert

Cutout 1.0— 1.10 p.m. 1.45 2.30 3.15 4.0 4.44

Exp. 3. Self-fert. G/fl, 3 cc. thick sperm, 10 cc. water 4 23 75 78 83

Cross-fert. if /.17, 1 cc. dilute sperm, 10 cc. water 100 100 100 100 100

Fert. Fert. Fert. Fert. Pert. Fert.

12 20 1.20 2 20 3.20 4'20 5'20'
Cut out 11.45-11. 55 am. (18°C.) (IVC.) (18°C.) (18° C.) (18°C.) {18-5°C.)
Exp. 4.

Self-fert. 7//, 3 cc. thick sperm, 10 cc. water... 0 <1<1 0 0 0

Cross-fert. J/t, 4 drops dilute sperm, lOcc. water 97 96 80 23 2 0

Fert. Fert. Fert. Fert. Fert.

12.20 1.20 2.20 3.20 420

Cut out 11.30-11.45a.m. (18°C.) (18°C.) (18-6°C.) (18-5-C.) (19° C.)
Exp. 5.

Self-fert. A'/fc, 4 cc. thick sperm, 10 cc. water ... 6 10 29 1 <1

Cross-fert. 7j/i, 2 drops dilute sperm, 10 cc. water 100 05 23 8 0

From the experiments already described on the length of time that
sperm and eggs remain capable of fertilizing, it appeared that the sperm
loses its capability rapidly, while the eggs "go off" much more slowly.
From this it would seem that the falling off of the cross-fertilization
percentages in Exps. 1, 4 and 5 is to be attributed to the failure of the
sperm rather than of the eggs.

From a comparison of the coiTCsponding self- and cross-fertilizations
in Exps. 1, 4 or 5 it seems that the self-fertilization percentages continue
to increase until the sperm begins to fail (as indicated by the decreasing
cross-fertilization percentages) after which they decrease again. In
Exp. 3, where the cross-fertilization percentages show that the sperm
did not commence to lose its fertilizing power during the progress of
the experiment, the proportions of eggs self-fertilized continued, to rise
steadily.

1 By "cut out" is meant tlie time at which the genital products were removed from the
duets.

" Tliis last fertilization showed much polyspermy, which may account for the irregu-
larity in the percentages. In all other cases recorded in this paper segmentation was
regular, unless otherwise mentioned.

2'SQ Sfifflirs in the Phi/siolof/i/ of Fertilization

III Exjis. 1, 4 and 5 the tempenitures of the water at thu moments
of fertilization are recorded. Table V shows that the maximum varia-
tion is only 1 C, and as there is no rise and fall correlated with the
rise and fall of the fertilization percentages, there can be no connection
between the two.

The conclusion can thus be drawn ft'om the experiments that lying

in water increases the self-fertilizing capacity of eggs and sperm. This

increase continues up to a maximum, after which it falls off again,

, probably due to the loss of both self- and cross-fertilizing power by the

sperm.

Although it is certain that there is an increase in the extent to
which self-fertilization takes place after the eggs and sperm have been
for some time in sea-water, yet the experiments do not decide whether
it is due to a change in the eggs or in the sperm. It was thought that
further experiments to decide this point could be made as follows :

About half the eggs contained in the oviduct are removed and
placed in sea-water. The hole in the oviduct wall through which the
eggs were removed is then clipped and the sperm removed from the
sperm duct to make up a suspension. At regular intervals equal
amounts of this sperm-suspension are used to fertilize (1) some of the
eggs already lying in water, and (2) some more eggs, just removed from
the oviduct. If the eggs (1) showed the typical rise and fiill in self-
fertilization percentages, while the eggs (2) did not, the phenomenon
would be due, at any rate in part, to an alteration in the eggs during
their stay in sea-water.

The experiment was trietl and was a comjjlete failure. The eggs
which had been for varying lengths of time in sea-water showed the
usual increase and decrease in fertilization percentages, but the propor-
tions in eggs taken from the oviduct immediately before each fertilization
were quite irregular. The reason for this will be apparent when the
results of experiments to be described below are seen; for it will be
shown that eggs and sperm taken from different parts of the genital
ducts of a given animal behave quite differently in their capacity for
self-fertilization. Now, whereas the eggs lying in water are thoroughl}'
mixed together before being used, and are thus homogeneous material,
every time that eggs are removed from the oviduct for comparison,
a sample is obtained having a ijuite different behaviour.

The reverse experiment, namely that of removing all the eggs and
half of the sperm from an animal and then taking samples of the
remainder of the sperm from the vas deferens at each fertilization, is

H. M. FucHS 237

impracticable for a further cause. It is impossible to make up sperm-
suspensions of identical concentrations each time that sperm is removed
from the duct.

Thus it cannot be decided whether the change in the capacity for
self-fertilization shown by the genital products on lying in water is due
to a change in the eggs or in the sperm.

3. Position of genital products in their' ducts.

The iiTegular variation in the self-fertilization percentages obtained
on successive days when an isolated animal is allowed to lay eggs and
sperm naturally must be explained in part at least by the differences in
the concentration of sperm, depending on the amount ejected on each
occasion. The importance of the sperm-concentration has already been
shown (pp. 232-233 above), but this may not be the whole reason for
the differences in the percentages just referred to. The phenomenon
suggests that the genital products of a given animal vary in their
capacity for self-fertilization from, day to day.

This hypothesis can be tested as follows. The eggs lying at the
inner end (base), in the middle and at the outer end (top) of the long
oviduct are removed separately from the animal and brought into sea-
water. The sperm is then removed from the sperm duct and used to
fertilize the three lots of eggs separately.

If equal amounts of the three lots of eggs showed the same amounts
of self-fertilization with equal concentrations of the sperm, it would
argue a uniformity of behaviour in one animal : although the possibility
would not be excluded that the eggs given off from the ovary at a later
occasion might act diffei-ently. If, on the other hand, the three lots
gave irregular differences in the percentages, it would be clearly shown
that eggs produced at one time have a different capacity foi- self-
fertilization from those produced at another time, since eggs lying at
the top of the oviduct are older than those lying at its base. Thei-e is
also a third possibility, namely that a regular gradation in percentages
from base to top would be found.

Experiments were made on this plan, and also the reverse ones of
testing samples of eggs, taken from the whole oviduct and well mixed
together, with sperm from different positions in the vas deferens. The
details of the first series of experiments were as follows :

The oviduct of an animal was clipped in one or more places and the
eggs removed separately from the different lengths thus divided off.
Comparative tests were made of the self-fertilizing capacities of these

238 Studies in the Phusiolofjjf of Fertilization,

different lots of eggs, iiiid dtlicr tests of their cross-fertilizing capacities.
For the former, sperm was removed from the sperm duct of the same
animal from which the eggs were derived, and a suspension of it made
up. Approximately equal quantities of the different lots of eggs were
put into di.shes containing 10 cc. of water, and to each 1 cc. of a thick
" own " sperm-suspension was added. For the cross-fertilizations a
dilute spcrm-susi)ension of another individual was made up. Fifty drops
of this were added to each of a number of dishes containing approxi-
mately equal amounts of the different lots of eggs in 10 cc. water. The
results of the experiments are shown in Table VI.

TABLE VI. (7.6.1 and 8.11.1.)

Vart of oviduct from wliich eggs were taken

Base Middle To|j

( J eggs selfed, Aja 4 — 12

( .-1 eggs crossed, Ajh lUO — 100

\ C eggs selfed, Cjc i 1 —

( C eggs crossed, Cjd 100 lUO —

E eggs selfed, Kje 9.i 90 —

E eggs crossed, £.'/.' 5 20 —

Exp. 4. G eggs selfed, (ijg 89 85 —

Exp. 1.
Exp. 2.
Exp. 3.

Part of oviduct from which eggs were taken

Exp.

Lower Upper

Base middle mituile Top

\ H eggs selfed, ///7i 0 0 0 0

^ K eggs crossed, Hji 00 IS 17 1

The Table shows at a glance that there is no uniformity in the self-
fertilization percentages of eggs taken from the base and from the top
of an oviduct of a given animal. Nor do the different animals agree in
the eggs from the top being always either more or less readily self-
fertilizable than those from the base. Exps. 3 and 5 show that the
same conclusions apply to the cros.s-fertilization percentages (in Exps. 1
and 2 an excess of sperm was present and no comparisons are possible).
Finally there is no correspondence between the self-fertilization per-
centages of eggs of a given animal, taken from the base and from the
top of the duct, and the cross-fertilization percentages of these eggs.
In Exp. 8 eggs from the base were more readily self-fertilizable than
those from the top, while the reverse held fir the cross-fertilizing
capacitii'S.

Besides the above, further experiments were laadu in which, not
only the eggs were removed separately from the different regions of the

H. M. FucHS

239

duct, but the sperm was treated similarly. It must be pointed out,
however, that comparisons between the effects of sperm from the base
and from the top of the vas deferens on any given lot of eggs are
necessarily very inexact. This is owing to the fact that it is impossible
to make up two different sperm-suspensions of exactly equal concentra-
tions, since the latter can only be judged by the eye, a comparatively
inaccurate method.

In each experiment the eggs were removed separately from the
different regions of the oviduct. Approximately equal quantities of
each of the lots of eggs were fertilized (1) by equal concentrations
of sperm from the base of the sperm duct, (2) by equal concentrations
of sperm from the top of the sperm duct, (3) by equal concentrations of
a sperm-suspension of another individual. Besides this, equal amounts
of the sperm-suspensions from the base and from the top of the sperm
duct were tested with " foreign " eggs. See Table VII.

TABLE VII. (12.16.1.)

Exp. 1. Eggs A selted with sperm from base of duct, AJoi
Egga .-1 selfed with sperm from top of duct, Aja-^
Eggs /I crossed, Ajm ... ...

Eggs C crossed with sperm «i from base of duct ...
Eggs G crossed with sperm a^ from top of duct ...

Part of oviduct from which
eggs were taken

Part of oviduct from which
eggs were taken

Base

6

U
97

(87)

Lower
middle

1

0

81

(87)

Exp. 2. Eggs B selfed witli sperm from base of duct, Bjhi
Eggs B selfed with sperm from top of duct, B/b.^
Eggs B crossed, Bjii

Eggs D crossed with sperm bi from base of duet
Eggs D crossed with sperm 62 from top of duct

Eggs E crossed with sperm &i from base of duct .
Eggs E crossed with sperm 62 from top of duct

These experiments are a further confirmation of those of Table VI
in showing the irregularity in behaviour of eggs from different parts of
an oviduct both in respect to self- and cross-fertilizing capacities — see

' The figures in brackets are tlie number of minutes after fertilization when the 4-eell
division was completed. See p. 245.

l^pper
middle

<1

0
85

(87)

"Jl

42

90
31

Top
<1

0

81

(87)

240 Studies in the Phusiohxjji of Fertilizatioti

Exp. 1, A/a„, Exp. 2, Bjbi, and B/n. The further point which these
two experiments were made to test, namely the behaviour of • sperm
from different regions of the vas deferens, must remain undecided for
the reason that the different suspensions cannot be made up to exactly
equal concentrations. In Exp. 1 sperm from the base failed to self-
fertilize, while that from the top fertilized the eggs of the same animal
to varjing degrees. In Exp. 2 exactly the reverse was the case.
Further, the tests of the two sperms in each ex)ieriment with "foreign"
eggs showed that the self-fertilizing powi'r went hand in hand with the
cross-fertilizing power. Whether, however, the behaviour of the two
sperm-suspensions means that there is a real difference between them
or whether it is due to a slight difference in the concentrations of the
suspensions must remain unsettled.

The final conclusion to be drawn from the experiments described in
this section is that eggs from different regions of the oviduct have quite
different self- (and cress-) fertilizing capacities, when treated with equal
concentrations of sperm. Whether or not there is a similar difference
in the behaviour of th^' sperm is undecided. The varying behaviour of
the eggs is in no way correlated with their position in the oviduct,
i.e. with their age, and we must therefore conclude that an animal
produces eggs which are at one time more and at another less prone to
self-fertilization.

IV, The Effect of x Sperm-Suspension on the Eggs

OF THE .S.\ME INDIVIDUAL.

As soon as it was found tiiat Cimut intentiixtli/y at Naples is not
totally self-sterile, but is self-fertile to a very varying degree in different
individuals, the proposed heredity experiments referred to in the
introduction had to be postponed until the limits and conditions of
self-fertility had been settled. In the preceding sections some of the
main factors have been described which influence the degree of self-
fertility, and although the latter is very variable, it has been shown that
a very much greater concentration of sperm is always necessary to
bring it about than is required to effect cross-fertilization. Moreover a
considerable proportion of individuals are always present, the eggs of
which cannot be self-fertilized at all.

The causes of this self-sterility have been investigated before, but
although the problem has been narrowed down considerably, they
remain fundamentally as obscure as ever. It was with a view to

H. M. FucHS •241

attacking tlie problem of the moans by which the immunity of eggs t(j
fertilization by " own " sperm is brought about that expei'iments on the
influence of egg-secretions on fertilization were begun. This investiga-
tion forms Part II of this paper. As the work progressed, so many
preliminary questions with regard to the effects of the egg-secretions
on normal cross-fertilizations in Ciona and other forms had to be settled,
that, at the time of writing, not enough experiments on the influence
of the secretions of " own " and " foreign " eggs on self-fertilization had
been made to justify publication.

In Part II it will be shown that the eggs of Ciona secrete a substance
into the water which stimulates spermatozoa, both of the same and of
other individuals, to effect cross-fertilization. Whether this secretion
fails to have the same effect on the self-fertilizing power of spermatozoa
of the same individual, or whether some other substance is secreted
which actually inhibits self-fertilization has not as yet been settled.
But whether such a possibilitj-' is true or not, there seems to be another
fiictor intimately connected with the difficulty of self-fertilization, and
that is a change brought about in the eggs by the presence of " own "
sperm. The experiments which will be described in this section seem
to show that if eggs be brought into contact with a sperm-suspension of
the same individual, their capability of being subsequently fertilized by
" foreign " spermatozoa is diminished as compared with that of eggs not
so treated.

The first series of experiments was carried out as follows. In each
instance the eggs of an individual were divided into two lots, which
were placed respectively in (1) plain water, (2) an opalescent suspension
of "own" sperm. At definite intervals 1 cc. of a "foreign" sperm-
suspension was pipetted into each of two dishes containing 22 cc. water.
The liquids so prepared were then poured separately on to (1) a drop
of eggs from the water, and (2) a drop of eggs fi-om the suspension of
"own" sperm. These fertilizations were repeated at intervals given in
Table VIII, so that the effects on the eggs of remaining for different
lengths of time in "own'' sperm could be compared.

The percentages of segmenting eggs were counted 80 minutes after
each fertilization, and at the same time the percentages of self-fertilized
eggs lying in "own" sperm-suspension (2) were counted. Table VIII
gives the results of the experiments.

It should be noted in the first place that the Table shows a decrease
in the percentages at each subsequent fertilization, a fact which, as has
already been pointed out, is almost certainly due to a gradual decline

Joiuii. of Geu. IV l(j

242 Studies in the Physiologn of Fertilization

in the fertilizing power of the sperm. The striking point in the results
is however that, for each pair of fertilizations, the percentages are

Time (
mixing €

Exp. 1. BIc

Exp. 2. Ajb

Exp. 3.

D/«

Exp. 4.

Fl9

Exp. 5.

Hji

Exp. 6.

Jjk

Exp. 7.

Ljm

TABLE VITI

. {\i.-21.Q

et sKq.)

ition, after
own" sperm

I'ercentage

fertilization

in egK8 from

water

Percentage

fertilization

in eggs from

"own " bpemi

Percentage
selfed

5 minutfs

1)7

84

<;1

25

100

97

2

45

100

95

3

65 „

96

87

3

85

94

9

3

105

72

66

3

125

47

38

3

145

21

14

3

165

6

5

3

' 10 minutes

100

100

—

30

100

100

—

50 „

100

95

10

70

98

58

13

90

51

89

16

130

6

20

i.e. all selfed)

20

( 5 minutes
(25 „

84

79

5

13

9

7

( 5 minutes

75

13

—

) 35 „

40

30

28

j 5 minutes

16

6

0

( 35

4

<1

<:1

I 5 minutes
( 35 „

17

9

0

5

«:1

0

S 10 minutes

90

68

0

Wo

0

0

0

lower with eggs which have been previously treated with "own" sperm
than with those not so treated. In most cases, moreover, the cross-
fertilization percentages of the former should be still lower than those
actually counted and recorded in the Table, since during their stay in
"own" sperm-suspension a certain number of the eggs had already been
self-fertilized. In order to allow for this factor the proportion of eggs
remaining in "own" sperm, which had been self-fertilized, was calculated
at the same time as the cross-fertilization percentages were counted.
These figures are given in the last column of the Table. In the last
fertilization of Exp. 2, the treated eggs show an apparently higher
cross-fertilization jDercentage than the untreated ; but on examining

H. M. FucHS 243

the last column it is seen that all these eggs had already been selt'ed.
Again in Exp. 4, although the treated eggs show for both fertilizations
lower percentages than the untreated, in the previously treated eggs
the second percentage is higher than the first. This is, however, really
due to the presence in the treated eggs of 28 '/^ already self-fertilized.

When the presence of self-fertilized eggs in the lots which have
been treated with "own" sperm is allowed for, the fall in cross-fertiliza-
tion percentages of the latter is even more marked than appeared at
first sight. This might be due to one of two causes. Either the
presence of "own" spermatozoa calls forth a reaction on the part of the
eggs hindering the entrance of the former, and also — although to a
much lesser degree — hindering the entrance of "foreign" spermatozoa:
or the presence of the small amount of sperm-suspension which is
necessarily carried over with the drop of eggs removed for cross-fertili-
zation itself inhibits the "foreign" spermatozoa. For spermatozoa in
the vas deferens are motionless, and this must be due either to the
absence of sea-water, or to the presence of a substance inhibiting move-
ment. If the latter exists, it must be present in a dilute form in the
suspension of "own" sperm, and might conceivably act on the "foreign"
spermatozoa when these are added to the drop of eggs taken from this
suspension. In order to test whether this is the explanation of the
diminished cross-fertilization pei'centages of the treated eggs, or whether
the latter have really been altered in their capacity for cross-fertilization
by their sojourn in "own" sperm, further experiments were made.
Before being cross-fertilized, the treated eggs were thoroughly washed
in a comparatively large volume of water.

As in the previous experiments, the eggs of each individual were
divided into two lots, and placed respectively in water and in opalescent
"own" sperm suspension. After a definite interval (10 mins. in Exps. 1
and 2; 15 mins. in Exp. 3) Ice. of "own" sperm containing eggs was
removed to 100 cc. of plain water in a finger-bowl, in which the eggs
were allowed to settle in order to remove excess of sperm. A definite
quantity of "foreign" sperm-suspension (given in Table IX) was pipetted
into each of two dishes containing 10 cc. water. These were then
poured on to separate approximately equal quantities of eggs (1) from
plain water, (2) from the finger-bowl.

In these experiments fertilizations were not made at different
intervals of time as in the former ones, but each was made double, two
different amounts of sperm being used. The results of the experiments
are recorded in Table IX.

i«— 2

244 Studies in the F/if/siolof/i/ of Fertilization

TABLE IX. (46.-2.7.)

Exp. 1. A'/o
Exp. 2. Pjq
Exp. 3. Ills

Eggs from Kkks from ' own "

water sptrm, washed

\ :i (iiojjs s)K'im ... 9.T 70

\ \ cc. spfiiii ... 100 100

( 3 drops sperm ... 3 <1

I 1 cc. spcnii ... II 4

( .H drops sperm ... 40 18

( 1 cc. sperm ... 82 64

The Table shows that the egg.s whicli have been in the presence of
"own" sperm behave in subsequent cross-fertilization in the same way
after the "own" sperm has been removed by washing as they did in the
previous experiments when this was not done. This makes it extreinely
probable that the real reason for the diminished capacity for cross-
fertilization is a change brought about in the eggs by the sperm of the
same individual.

An examination of Tables VIII and IX .shows that the change in
the eggs, as indicated by the lowered cross-fertilization percentages, is
comparatively small, and that when an excess of "foreign" sperm is
present, as in Table VIII, Exp. 2, and Table IX, Exp. 1, 1007„ of these
eggs can be fertilized. That the eggs can be cross-fertilized after the
treatment, although with rather less ease, does not mean that there
may not have been a large change in them, as regards their receptivity
to their "own" sperm. Whether this is so or not cannot be tested, as
our only criterion is that of comparing the extent to which they can be
cross-fertilized, with and without the previous treatment. The facts
certainly point, however, to the sperm having caused an alteration in
the eggs of the same individual, and to this alteration being at any rate
one of the means by which self-fertilization is effected.

V. Comparison of the Subsequent Development of Eggs self-

AND cross-fertilized UNDER VARIOUS CONDITIONS.

1. Eggs cross-fertilized at different intervals after the reiiiuvul of
the genital products from the body of the animal.

The foregoing investigations, concerning some of the conditions
which favour and limit the extent of self-fertilization, and the reasons
for the self-sterility itself bring the main part of this division of the
work to a conclusion. A considerable number of observations were,
however, also made on the subsequent development of eggs self- and

H. M. FucHS 245

cross-fertilized under different conditions. It was thougiit that these
experiments were worth recording, and the details, together with the
conclusions to be drawn from them, are given in the following sections.

The first series of observations concerns the comparative rates of
development and the condition of the larvae hatched out when different
lots of eggs are cross-fertilized at regular intervals after the genital
products have been removed from the body of the animal into sea-water.
The fertilizations were made in exactly the same way as those described
on p. 234 above, and indeed several of the experiments in this section
are identical with those recorded in Table V, the subsequent develop-
ment of the eggs having been noted. The method it will be recalled,
was briefly as follows. P^or each experiment eggs were removed from
one individual and sperm from another. At definite intervals (given in
the Table below) approximately equal quantities of the eggs were fer-
tilized by the addition each time of exactly equal amounts of the dilute
sperm-suspension.

The subsequent observations were as follows :

III the first place, the percentages of eggs fertilized were counted as
usual.

Secondly, the rates of segmentation of the eggs were compared by
noting the length of time after fertilization at which the 4-cell division
took place. This stage was fixed upon as it is the easiest to observe
rapidly and accurately under a low power of the microscope. Now,
although all the eggs in a given lot do not complete the 4-cell division
at a given moment, yet in general the majority divide almost simul-
taneously, and for this reason the criterion adopted for the comparison
of the rates of segmentation was the time at which the first few eggs in
a given sample were seen to have completed their second division. This
naturally involves a certain latitude of experimental error, and in con-
sequence the figures given in the Tables below are not correct to a
minute. The extent of this error, however, does not exceed two minutes,
and it will be seen that the differences between the times taken to
complete the second divisions in the different fertilizations in a given
experiment are usually considerably larger than this.

The rapidities of development of the embryos up to hatching were
compared by noting the lengths of time after fertilization at which the
first larvae emerged from the eggs in the different dishes. It is usually
about half an hour after the first has come out before the majority have
emerged from the eggs, and when the larvae are weakly, this time is
considerably longer. The comparisons were made by observing when

246 Studies in the Phji.vology of Fertilhntioii

the first larva of each lot hatched out. In the cx})criiiieiits in wliich
the times of hatching were notefl tlie conrlitions of the resulting- larvae
were also recorded.

The figures for these experiments are given in Table X.

TABLE X.

ExplanatidH of Tnhli\

"Time of fertil." The approximate time in liouis, after the genital products had been
removed from the body of the animal, at which the fertilization was made.

"Percent, fertil." Percentage of eggs fertilized.

"Time of 4-cells." Time in minute.-i after fertilization at which the first fonr-ccll division
was completed.

"Time of hatching." Time in hours and minutes after fertilization at whieh the first
larvae hatched out of the eggs.

Time of
fertil.

Percent,
fertil.

Time of
4-cells

Time of
hatchini,'

Condition
of larvae

Series A. (9.13.1.)

1

100

115

22, 8

Good

(Temp. 19 -.5— 21-5=^0.)

■J

100

108

22, 3

Good

Exp. 1. .-I/m

3

100

101

22, 20

Good

.

4

100

100

23

Fair

f

1

100

no

22, 48

V. weak, few hatched

Exp. 2. C/m

2

100

98

21, 43

Good

4

100

95

22, 10

V.weak, fewhatohed,
malformed

{

1

100

113

22, 48

V. weak, few hatched

Exp. 3. FIc

2

100

98

22, 28

Good

( = Table V, Exp. 2, Fjc)

4

100

95

21, 40

Good

f

1

100

108

—

Eip. 4. Bjm

1
1

■>

100

98

—

.

1

4

100

95

—

—

Time of
fertil.

Percent,
fertil.

Time (
4-cells

3f

Series B. (11. l.").!.)

1 ''

90

105

Exp. '..

./

/«, -

25

100

[ 2.i

-^ 1

(88)

1

100

99

Exp. C.

(,7« •

If

100
100

81

I Hi

1 -f

100
93

90
97

Eip. 7.

a

/" -

If,

19

5

100

100

98
89
90
91

Exp. 8.

Hh -

■■i'i

100

80

( = Table III, Exp. 8. .

fJIfi)

■i

100

87

H

100

76

H.

M. FucHS

s

TABLE

X {co7itinned).

Time of
fertil.

Temp.

Percent.
fertU.

Time of
4 -cells

Series C. (13.17.1.)

' 1

18° C.

100

82

2

do.

100

86

Exp. 9. Aim

S

do.

100

87

4

do.

85

88

■, 5

do.

3

93

' 1
2

18° C.

96

83

Exp. 10. C'/a -

do.

3

86

3

do.

<1

(92)

1 i

18° C.

100

84

14

do.

100

86

Exp. 11. J/o -

24

do.

100

85

34

do.

84

89

^ H

do.

12

88

f 4

18° C.

97

78

1*

do.

96

85

Exp. 12. Jji -

24

do.

80

87

{ = TableV, Exp. 4. Jji)

H

do.

23

88

I 44

do.

2

87

Series D. (14.18.1.)

f ^i

18° C.

100

88

Exp. 1.3. B/m <

3i

do.

18 -5° C.

78
16

!)1
91

I 4J

do.

4

86

Exp. 14. DIh \

- H

18° C.

4

87

2*

do.

15

89

■ 3i

18-5° C.

<1

(95)

247

Considering first the four experiments forming Series A, it will be
noticed that in each case the eggs fertilized later completed the 4-cell
division in a shorter time than those fertilized earlier. Thus in Exp. 1,
eggs fertilized one hour after being removed fi-om the animal into sea-
water took 115 minutes to complete the 4-cell division, those fertilized
after two hours in sea- water took 108 minutes, those after three hours
101 minutes, and those after four hours only 100 minutes. The figures
for the other three experiments show a similar increase in rate. This
point has already been recorded by Morgan (9), although he made no
exact observations on the times of segmentation. With regard to the
later development, the hatching rates of this series show that the
second fertilization continued to have a more rapid rate than the first
ones. In Exps. 1 and 2, the parallel with the earlier segmentation
ceased here, for in the latest fertilized eggs the rate of development
of the embiyo slowed down again. In Exp. 3, on the other hand, the

248 StitfJirs ill the PhjiHioIiKin of Fcrtilizatioii

eggs fertilized after ;i previous stay of four lioiirs in srawater hatched
quickest of all, just as the 4-cell division of these eggs had been com-
pleted in the shortest period.

In comparing the last column of Table X, whieJi givi's the condition
of the larvae, with that in which the times of hatching are recorded, it
is seen that those larvae which emerge from the c^gg in the shortest
time from fertilization are the healthiest.

In the four experiments of Series B the i-ates of early segmentation
were noted, but not the times of hatching. The general result is th('
same as that of Series A, namely that the later fertilized eggs segment
quicker, but Exp. 8 shows a marked irregularity difficult to account for.
There is a fall from the 91 minutes of the 1^-hour eggs to 80 minutes
of the 2|-hour eggs, after which the next fertilized lot give 87 minutes,
and then the final batch drop to 76 minutes again. Finally in Exp. 6,
there is a decrease in the times at which the 4-cell stage was attained
from the 1-hour lot (99 minutes) to the 2^-hour lot (81 minutes), after
which there is a rise to 90 minutes again. This is a phenomenon
similar to the change in the hatching rates in Series A. The segmen-
tation time of the last fertilization in Exp. 5 is given in brackets, as it
is not really justifiable to compare this observation, made on the very
few eggs that were fertilized in this case, with the large numbers in the
other batches.

Exps. 5 and 7 show that the increasi' of segmentation rates with
succeeding fertilizations went hand in hand with a decrease in the per-
centages of eggs fertilized.

The experiments of Series C show an exactly reverse phenomenon
to those of Series A and B. In every case the segmentation rates
become slinver with successive fertilizations. This is especially marked
in Exp. 9, where there was a gi-adual rise from 82 minutes of the 1-hour
fertilization to 93 minutes of the 5-hour lot. The two experiments
forming Series D behaved similarly, although there was a final increase
in the rate for the last fertilization of Exp). 13.

It was thought that there might be a connection between the
change in temperature during the progress of the experiments and the
alterations in the rate of segmentation. The evidence, however, seems
to point rather against this. In Series A the temj)erature rose from
19*5" to 21-5° C. during the experiments. In Series C it remained
constant, and in Series D rose from 18 to 18'5"C. If the rise of two
degrees in the experiments of Series A will account for the increase in
the segmentation rates, there is no fall in temperature in the later

H. M. FiTCHS J+O

series correlated with the decrease in th(> latter. Moreover in the first
series, although thi' segmentation became more rapid in later fertili-
zations, the time taken to complete the 4-cell division was longer than
that of the slowest segmentation in Series C. Nevertheless the tem-
perature during the latter was throughout lower than during the fomner
series of experiments. The possibility is, however, not excluded, and
unfortunately time would not allow of a fiirther investigation. Never-
theless the difference in behaviour of the eggs would seem to depend
rather on some condition of the animals from which they are taken.
The four series were made on four different days, and each with a
different batch of animals brought into the laboratory. This, taken
together with the fact that the experiments of each series agreed in
general with one another, seems to support this view.

2. Eg(js and sperm from different parts of genital ducts.
During the experiments made to investigate the effect of the " age "

of eggs and sperm, as indicated by their relative position in the genital
ducts, on the extent of self- and cross-fertilization, the segmentation
rates of the fertilized eggs were noted. The lengths of time after
fertilization at which the first 4-cell divisions took place are recorded in
Table VII ( p. 239) for some of the lots of eggs.

A comparison between the times taken by 6* eggs cross-fertilized :
(1) by sperm a, from the base of the sperm duct, and (2) by sperm a„
from the top of the sperm duct, in Exp. 1, shows that the segmentation
rates were identical, although the percentages of eggs fertilized were
very different in the two cases. Again, eggs from (1) base, (2) middle,
and (3) top of the oviduct cross-fertilized by the same sperm, all segment
at the same rate, although the jiercentages fertilized may be different.
This is shown by the segmentation times in Exp. 1, cross Ajm and
Exp. 2, cross Bjn of Table VII.

Thus, neither the position in the genital duct of the sperm used to
make a cross, nor that of the eggs crossed causes any variation in the
early rate of division of the segmenting eggs.

3. Gojnparison of development after self- and cross-fertilization.

The first experiment consisted in a comparison of the segmentation
rates of (1) eggs self-fertilized (Kjk), (2) eggs of the same individual
cross-fertilized (K/p), and (3) eggs of another individual cross-fertilized
by the same sperm as that used for the self-fertilization {L(k). In each
case the eggs were fertilized at four different intervals after the genital

250 ShidieH hi the Phjiaiolofiji of Fertilization

products had been removetl into the sea-water. The details and results
of the experiment are recorded in Table XI, and it will be noticed that
the experiment is the same as that given in Table V, Exp. 5. In
addition, the cross Kjp was made, and is tabulated here only, as the
percentages of eggs fertilized were immaterial to the matter under
discussion in the previous section.

TABLE XI. (14.18.1.)

For explanation of headings of Colamns see Table X.

Eggs A', croes-fertilized, Kjp

Self-fertilization, Kjh

Sperm k, cross-fertilized, Ljk

rime of
fertil.

Temp.

Percent.
fertiL

Time of
4-ceUs

i

18° C.

100

90

If

do.

86

92

2i

18-5° C.

30

92

3?

do.

7

91

?

18'- C.

6

87

If

do.

10

87

n

18-5° C.

29

87

n

do.

1

91

1

18° C.

100

87

If

do.

65

87

2|

18-5° C.

23

87

35

do.

8

85

The variations in the segmentation times of the eggs fertilized at
different intervals in any of the three crosses were small. It is seen by
comparing Kjk with Ljk that there is practically no difference in the
rate of early segmentation of eggs self- and cross-fertilized. The four
fertilizations Kjp took rather longer to divide than the other two com-
binations. The results further emphasize the fact that the rates of
segmentation do not depend on the percentages of eggs fertilized.

As was explained in the Introduction, the original scheme of work
has not yet been carried out, namely that of rearing reciprocal cross-
fertilized families to maturity, in order to find the degi'ee of cross-
fertility of sisters inter se and of members of one family with those of
the reciprocal. This has not yet been attempted owing to the many
preliminaries to be settled first, the investigation of which forms the
substance of this paper. A number of families were, however, reared,
partly in order to settle the optimum conditions of food, etc., (G), but
more especially to compare the later development of animals derived
from cross- and from self-fertilized eggs. The details of two t3'pical
experiments are given below.

H. M. FiTCHS 251

It should be noticed in the first place that, whereas in all previous
experiments in cross-fertilization very dilute sperm-suspensions were
used so that the percentages of eggs fertilized should lie between 0 and
100, thus allowing of comparison, the fertilizations in the following cases
were all made with an excess of sperm. The reason for this was, of
course, that the object of the experiments was not to compare per-
centages of eggs fertilized under different conditions, but to obtain the
maximum number (100°/,_,) of developing eggs for rearing purposes.

In the first experiment (Table XII) A eggs were self-fertilized (A/a)
and B eggs were self-fertilized (B/b). At the same time the reciprocal
cross-fertilizations were made between these two individuals (A/b and
B/a) and eggs of a third individual G were cross-fertilized with a
sperm (G/a).

TABLE XII. (15.20.1.)

Percent. Time of Time of Settled Alive, Alive,

fertiL 4-cells hatching down 8 days old 20 days old

„ ,, , . f Ala 7 86 19, 37 V. few None —

Self-fert. ... ' ' ,,

\ Bib < 1 83 19, 30 None — —

^ Ajb 100 83 19, 15 Most / Equal number , Fevfest of Ajb,

Cross-fert. \ Bja 100 83 19,27 Many J of .-1/6 and B/a, | medium num-

\ Gja 100 86 19, 26 Most i more of C/a \ ber Bja, most

'^ Cja

The Table shows that the excess of sperm gave in each cross-fertili-
zation 100°/'(, of segmenting eggs. The second column of the Table
shows that there was very little difference in the early segmentation
rates of the five fertiliza.tions. Both when b sperm was used to self-
fertilize B eggs and to cross A eggs, the 4-ceU stage was reached at the
same time — 83 minutes. Again, when a sperm was used to self A eggs
and to cross G eggs the segmentation rates were identical (in this case
86 minutes for 4-cell stage), but when the a sperm fertilized B eggs the
latter segmented a little quicker (83 minutes).

Thus the early segmentation was not slower in the self-fertilized
eggs than in the cross-fertilized, but the times of hatching (given in the
Table in hours and minutes after fertilization) show that the segmenta-
tion rate of the former slowed down a little in the later stages.

In the column headed "Settled down" is given the proportion of
larvae in each case which fixed themselves after their brief free-
swimming period. The proportions were roughly estimated by eye —
the only method practicable — and are independent of the absolute
. numbers of larvae pi-esent in the different cultures, which varies, of
course, with the percentages of eggs fertilized. It is striking that none

252 Studies in the Plijisiolofiji of Fertilization

of the few larvae present in the Bjb culture settled down, and very few
in the Aja, whereas a large proportion fixed themselves in the cross-
fertilized lots. The next examination was made eight days afterwards,
when no individuals derived from self-fertilized c'ggs were found to have
survived, while those from cross-fertilizations were growing rapidly.

The second experiment, made on the same lines as the first, is
recorded in Table XIII.

TABLE XIII. (15.22.1.)

Percent.

fertil.

Time of
4 -cells

Time of

hatcliiriK

Condition of
larvae

8 cIaj-8 old

20 days old

Self-fert.

ICjc

^1
41

81

21, 48

Good, but not

so vigorous as

crosses

One or two

alive

No survivors

(BIc

100

82

21, 29

V. good

Many alive

Many alive

Cross-fert.

Clb

100

82

21, 31

V. good

Many alive

Many alive

Cja

100

80

21, 39

V. good

Many alive

Few alive

.Ale

100

80

21, 19

V. good

Many alive

Few alive

In this case, of the self-fertilizations only C/c gave enough seg-
menting eggs to make observations on. The rate of early segmentation
was here slightly slower than in the crosses, and the time of hatching
was considerably later. Moreover the G/c larvae hatched out were not
so vigorous as those of the crosses, which were about equal to one
another in this respect. Eight days later one or two only of the self-
fertilized individuals which had fixed themselves were surviving, and
after 20 days these were found to have died ofi".

It should be mentioned here that after the majority of the larvae
had settled down in their different dishes, the latter were washed in a
stream of sea-water to remove all larvae which had failed to fix them-
selves together with all that had settled on the surface-film'. The
dished were then sunk in a large tank of water, so that the different
cultures should be exposed to identical conditions during the growth of
the young animals.

In the last column of Table XIII it will be noticed that after
20 days fewer of the crosses G/a and A/c were living than of B/c and
C/fc. Nevertheless, the former had shown a slighth' quicker rate of
early segmentation than the latter, and the time of hatching o{ A/c had

' In later experiments, where a strict comparison between the states of development
in the various cultures was not wanted, the animals which settled on the surface-film were
not discarded. They were indeed in most cases found to grow more rapiilly than tliose on
the walls of the dishes.

H. M. FucHS 253

been the earliest in the crosses, although that of Cja had been the
latest. There seems to be little or no correspondence between the
relative rapidity of segmentation and the subsequent development.

The larvae of each cross which settled down on the first, on the
second and on the third day after hatching were reared separately.
Those from the first and second days developed equally well, but those
from the third much worse in each case.

Besides the two experiments given in Tables XII and XIII, a con-
siderable number of other similar ones were carried out. All agreed in
showing that the early segmentation of self-fertilized eggs is little if at
all slower than of cross-fertilized, but that the larvae of the former
hatch out somewhat later. Very frequently, however, the larvae from
the self-fertilizations do not fix themselves at all, but die off. When
they do settle down they fail to develop further, perishing in the course
of a few days. One exception only was found to the last statement.
A self-fertilized culture {Aja) made on June 20th gave a low percentage
of fertilization. Some of the larvae fixed themselves, and on June 22nd
— more than a month later — four individuals were still alive. They
had attained the length of 1 cm. and appeared to be very healthy.
Young from both of the cross-fertilizations, Ajh and Bjh, however, grew
nuich moi'e rapidly.

VI. Summary of Experimental Results.

(1) A greater concentration of sperm is usually necessary to bring
about any self-fertilization than would cross-fertilize 100°/^ of "foreigTi"
eggs.

(2) An increase in the concentration of the sperm-suspension causes
an increase in the number of eggs self-fertilized.

(3) The proportion of eggs self-fertilized increases with the length
of time the eggs and sperm have been in sea-water before fertilization
is effected. The percentages rise to a maximum and then decrease
again, the time of the maximum being different for each individual.
The subsequent decrease in the percentages is probably due to a falling
off of the "fertilizing power" of the sperm-suspension. It cannot be
determined whether the rise in self-fertilization percentages is due to a
change in the eggs or in the sperm. The cross-fertilization percentages
decrease as the time the eggs and sperm lie in sea-water before fertili-
zation increases.

254 Studies in the PhiisioUxjij of Fertilization

(4) There is no ui)iJbriiiit,y in the fertilization percentages of eggs
from different parts of an oviduct, fertilized with equal amounts of a
given sperm-suspension. Sometimes eggs from the outer end, at others
eggs from the inner end are more readily self-fertilizable. Nor is there
any correspondence between the relative ease of self-fertilization and
that of cross-fertilization of eggs from different parts of the duct.
Pnjbably the same applies to sperm t;iken from different regions of the
vas deferens. Thus the degree to which eggs of a given individual can
be self-fertilized varies with each batch produced.

(5) Contact with a suspen.sion of "own" spca-ni decreases the ease
with which eggs can subsequently be cross-fertilized.

(()) The rates of segmentation of eggs cross-fertilized at increasing
lengths of time after the eggs and sperm have been brought into sea-
water either increase or decrease.

(7) Cross-fertilized eggs from different regions of the oviduct seg-
ment at the same rates. This is also true for eggs cross-fertilized by
sperm from different parts of a vas deferens.

(8) The rates of segmentation are independent of the percentages
of eggs self- or cross-fertilized.

(9) The early segmentation lute is not slower in self- than in cross-
fertilized eggs, but the former hatch out a little later. Many of the
larvae from the self-fertilizations fail to settle down, and those which do
almost always die off in the course of a few days.

(10) There is little or no relation between the relative rapidities of
segmentation and the stdisequent developnu'nt of different lots of cross-
fertilized eggs. In a given cultuiv, however, the larvae which .settle
down last develop worst.

VII. Conclusion.

In comparing Castle's and Morgan's work with the present results,
it is evident that there are races of Ciona intestinaUs which differ con-
siderably with reganl to their capacity for self-fertilization. This is not
exactly the same phenomenon as that originally discoveivd by Darwin
in Reseda and recently reinvestigated from a hereditary standpoint by
Compton (2). Here there are individuals which are completely self-
sterile and others which are completely self-fertile. Compton's work is
not as yet finished, but the results so fir obtaiiii'd indicate that in

H. M. FucHS 255

Reseda self-fertility is a Mendelian dominant to self-sterility. For
Clona, however, in those individuals where self-fertilization can most
easily be brought about, it takes place to a very much lesser degree
than does cross-fertilization. A much greater concentration of sperm is
needed to induce a comparatively high percentage of self-fertilized eggs
than would easily cross-fertilize all the eggs of another individual. On
the other hand many individuals were experimented upon at Naples in
which no eggs at all could be fertilized with sperm from the same
animal, although in general the Naples Ciona seems to be much more
self-fertile than the races used by Morgan. Again, in Reseda apparently
an individual is either self-fertile or self- sterile. This is not at all the
case with Ciona at Naples, where a given animal may vary widely in
its capacity for self-fertilization with each lot of eggs and sperm
produced'.

The question arises as to whether the sterility of hermaphrodite
animals is confined to the genital products of the same individual;
whether all individuals are equally fertile when crossed with one another,
presupposing of course that the eggs and sperm are mature and in good
condition. Morgan attempted to investigate this question in Ciona and
concluded that all individuals are not equally fertile inter se. A discus-
sion of Morgan's methods has already been given (Section II) and it is
considered that his conclusions are unjustified. The question is not an
easy one to attack owing to the difficulty of making different sj^erm-
suspensions of nearly equal concentrations ; for a small difference in the
strength of a sperm-suspension makes a large difference in the pr(_)-
portion of eggs cross-fertilized. Nevertheless if there be considerable
variations in the degree of cross-fertility, variations at all comparable
with the extent of self-sterility, they would certainly be detected. It
was not attempted to investigate the question in the present work, but
it can be stated that in practically every case 100°/^ of segmenting eggs
could be obtained as a result of cross-fertilization, provided that enough
sperm was used. There were of course a few cases in which some of
the eggs were obviously pathological and could therefore not be fer-
tilized, but this does not affect the question. It is possible that the
degree of cross-fertility may be much less in nearly related individuals
(cf. Correns (4)), but this naturally cannot be decided with material
taken from the sea. It is hoped that the laboratory cultures now being
reared will shed further light on the subject.

' This may have been somethhig like the same phenomeuou ia Correus' Cardaiiiiite,
for repeated poUmatious with the same plant did not alv^ays give the same result.

256 SfncUes iu the P/ij/s'iolo(/i/ of l-'ertilizatioii,

The fact that self-fertilization can usually only bi' brought about at all
in concentrated sperm-suspension suggests that in the water containing
the crowded spermatozoa there may be a substance which favours the
self-fertilization. This cannot be connected with the reaction of the
water, however, as the following test shows. A milky suspension of
Ciona sperm was filtered. Five minutes after the suspension had been
made the filtrate gave the same reaction as normal sea-water with
neutral red and with phenolphthalein. An hour and a half afterwards
the latter indicator showed that a freshly filtered sample of the same
thick suspension was very slightly more acid than normal sea-water-
But, as will be shown in Part II, acid diminishes, not increases, the
fertilizing power of a sperm-suspension. Experiments also will be made
in the following way to test whether the thick sperm-suspension contains
substances which favour fertilization. A concentrated sperm-suspension
will be made up and then filtered through a porcelain filter which keeps
back spermatozoa. Exact comparative experiments will then be made
to compare the effect of this filtrate with that of ordinary sea- water on
the proportion of eggs fertilized by a definite concentration of sperm.
It may be, however, that self-fertilization takes place only with con-
centrated sperm-suspensions because only one .spermatozoon out of a
very large number has the power of fertilizing eggs derived from the
same individual.

The usual (though not invariable) progressive increase in capacity
for self-fertilization shown by eggs and spermatozoa as they lie in sea-
water suggests a connection with the maturation of the eggs. Golski (7)
has shown that in the unfertilized egg of Ciona the first maturation
spindle is already formed, but the first polar body is not given off until
the spermatozoon has entered'. It might be that the increase in self-
fertility is connected with the preparation for the first maturation
division. At all events some change must come about in the eggs or
spermatozoa or both after thty have bi'cn shi-d into tht- sea-water. It
is very interesting to note in this coiuiection that self-fertilization can
only take place to a very small extent in nature. For the only time
that the eggs are in the presence of concentrated sperm of the same
individual is for the few moments alter the genital products have been
exuded from the ducts beibre they are ejected through the atrial
aperture: but at this time the eggs are usually not yet in a condition to
allow of any si'lf-fertilization. When tjioy hav(> reached this condition

' I have not been able to obtain a copy of (loLsUi's iiaiHT, but beliew this .statement to
be correct.

H. M. FucHS 257

some time after having been deposited, the eggs are no longer in
the presence of the concentrated "own" sperm, which has become
diffused in the surrounding water and washed about by waves and
currents. In allowing an animal to deposit eggs and sperm when
isolated in a small dish, the conditions are quite otherwise, for the
water into which the eggs are deposited becomes at the same time
charged with spermatozoa. The latter can subsequently self-fertilize
the eggs, to a greater or less extent according to concentration and
individual capacity, when the optimum time has been reached.

The cause of self-sterility is hardly touched upon in this investi-
gation. The search for a cause led to the investigation of substances
secreted by eggs into sea-water. The results of this work form the
substance of a separate Part, which, however, deals with cross-fertili-
zation alone. So many points had to be settled with regard to the
effects of the egg-secretions on spermatozoa used to effect ordinary
cross-fertilization in Giona and other forms, that hardly any self-fertili-
zation experiments had been made. It is therefore really premature to
discuss possible means by which self-sterility is brought about. The
experiments detailed in Section IV above indicate, however, that one of
the factors may be a change set up in the egg by contact with sperma-
tozoa of the same individual — a change which inhibits the entrance of
such spermatozoa, and also, although to a much lesser degree, hinders
the entrance of " foreign " spermatozoa.

As is well known, Darwin (5) first made a thorough investigation of
the effects of self-fertilization on the offspring, showing that in flowering
plants such offspring are not in general so vigorous as those derived
from cross-fertilization. In Giona this effect of self-fertilization was
veiy striking. As has been shown above, the early segmentation is not
usually slower in self- than in cross-fertilized eggs. It is interesting to
note also that there is no difference in the segmentation rates of self-
fertilized eggs from different parts of an oviduct, although the various
lots may show quite different capacities for self-fertilization. The later
segmentation of self-fertilized eggs is usually a little slower than that
of cross-fertilized. In a few cases the larvae failed to hatch well, that
is to say, they had difficulty in breaking out of the egg membranes, but
this was not usually the case. In many cultures derived from self-
fertilizations the larvae failed to settle down after their free-swimming
period, but in most instances they settled down in the same way as
those from cross-fertilizations. It was after the settling down and
metamorphosis, however, that the marked effect usually showed itself.

Journ. of Gen. iv 17

258 Studies in the P/iysiolof/i/ of Fertilization

With one exception, in which four individuals were reared for over a
month, in all the cultures derived from self-fertilizations the young
metamorphosed animals died off during the first week. This was very
striking in contrast with cultures from cross-fertilizations in which the
animals grew rapidly from metamorphosis onwards. Potts (13) states
that " The pathological development which Castle found characteristic
of self- fertilized embryos did not occur in my experiments." It is plain,
however, that the effect of self-fertilization does not usually manifest
itself in the embryo, but at, or more usually after, metamorphosis.

REFERENCES.

1. Castle, W. E. "The Early Embryology of Ciona intestinalis." Bull. Mus.

Comp. Zool., Harvard, Vol. xxvn. 1896, p. 203.

2. CoMPTON, R. H. " Preliminary Note on the Inheritance of Self-.sterility in

Reseda odorata." Proc. Camb. Phil. Soc. Vol. xvii. 1912.

3. " Phenomena and Problems of Self-sterility." A^ew Ph/tol. Vol. xil.

1913, p. 197.

4. CoRRBKS, C. "Selbststerilitiit imd ludividualstoffe." Festschr. d. med.-nat.

Ges. z. 84, Versanmil. deutsch. Naturf. u. Arzte, MUnster, 1912.

5. Darwin, C. The Effects of Cross- and Self-fertilization, 1876.

6. FncHS, H. M. "The Effects of Abundant Food on the Growth of Young

Ciona intestinalis." Biol. Centralbl. Bd. xxxiv. 1914, S. 429.

7. GoLSKi, St. " Reifung und Befruchtung de.s Eies von Ciona intestinalis,"

Bull. Acad. Cracovie, 1899, p. 124.
7a. GuTHERZ, S. " Selbst- und Kreuzbefruchtmig bei solitaren Ascidien." Arch,
f. mikr. Anat. Bd. lxiv. 1904, S. 111.

8. Hartmeyer, R. Fauna Arctica, Bd. ni. 1904, S. 301.

9. Morgan, T. H. " Self-Fertilization Induced by Artificial Means." Journ.

E.vp. Zool. Vol. I. 1914, 1). 13,5.

10. " Some Further E.xperiment-s in Self- Fertilization in Ciona." Biol. Bull.

Vol. VIII. 1905, p. 313.

11. "Cross- and Self-Fertilization in Ciona intestinalis." Arch. f. Entw.-

Mech. Bd. xxx. 1910, S. 206. (Festschrift.)

12. Van Name, W. G. "SimjJe Ascidians of the Coasts of New England."

Proc. Bost. Soc. Nat. Hist. Vol. xxxiv. p. 439.

13. Potts, F. A. "Notes on Fi-ee-Living Nematodes." Quart. Journ. Micr. Sci.

Vol. LV. 1910, p. 481.

14. Seeliger, 0. "Tunicata" in Bronns Tierreich, Bd. in.

H. M. FucHS 259

PART II. THE ACTION OF EGG-SECRETIONS ON THE
FERTILIZING POWER OF SPERM.

CONTENTS.

PAGE

I. Introduction 260

II. Methods 261

III. Effects of egg- and ovai'y-extracts on fertilization percentages . . . 264

1. Cross-fertilization of Ciona in egg-extract 264

2. Cross-fertilization of Ciona in boiled egg-extract .... 266

3. Cross-fertilization of Ascidia in egg-extract 266

4. Cross-fertilization of Ciona in ovary-extract ..... 267

5. Comparison of the effects of egg- and ovary-extracts on fertilization

in Ciona ........... 268

6. Comparison of the effects of "foreign" and "own" extracts on

fertilization in Ciona ......... 268

IV. Does the extract affect the eggs or the sperm ? 270

1. Effect of extract on the sperm of Ciona ...... 270

2. Effect of extract on the eggs of Ciona 272

3. Effect of extract on the fertilizing power of the sperm of Strongy-

locentrotus ........... 273

V. Effects of "egg- waters" on the fertilizing power of sperm .... 276

1. Experiments with Ciona 277

2. Experiments with Arbacia ........ 277

3. Experiments with Strongylocentrotus 278

VI. Effect of Ciona blood on the fertilizing power of Ciona sperm . . . 279

VII. Influence of "egg-waters " and egg-extracts of other animals on the fertilizing

power of Ciona sperm 282

1. Phallusia egg-extract 282

2. Arbacia egg-extract 282

3. Arbacia egg-water 283

4. Strongylocentrotus egg-extract 284

5. Strongylocentrotus egg-water 285

Vin. Further experiments to test the specificity of the egg-secretions . . . 287

1. Comparison of the effects of Strongylocentrotus and of Ciona egg-

extracts en the fertilizing power of Strongylocentrotus sperm . 287

2. Comparison of the effects of Strongylocentrotus and Sphaerechinus

egg-extracts on the fertilizing power of Strongylocentrotus sperm 288

3. Comparison of the effects of Strongylocentrotus and Echinus egg-

extracts on the fertilizing power of Strongylocentrotus sperm . 288

4. Comparison of the effects of Strongylocentrotus and Echinus egg-

extracts on the fertilizing power of Echinus sperm . . . 289

17—2

260 Studies in the Physiology of Fertilization

PACE

5. Comparison of the effects of Stron/iiilocenlrotus and EchimiK eg;,'-

extracts on the fertilizing power of Strungylocentrotus sperm used

to fertilize Echinus eggs 289

6. Comparison of the effects of Strongrjloeentrntus and Asleria^ egg-

extracts on the fertilizing power of Strongiilocentrolus sperm . 291

7. Comparison of the effects of Strotipyloceiilroliif and Asterias egg-

waters on the fertilizing power of Strongylocentrotus sperm . 292

IX. Effects of the H-ion concentration of the water on the fertilizing power of

sperm ............ 293

1. Effect of a decrease in H-ion concentration ..... 293

2. Effect of an increase in Hion concentration 294

8. The reactions of Ciona and -■l-stcrins egg-extracts and of Cioiia blood 296
X. Conclusion 298

XI. Summary of experimental results 300

References 301

I. Introduction.

The investigations described in this paper arose out of experiments
on self-fertilization in Ciona intestinalis, an account of which is given in
the previous part of this paper. The present experiments were carried
out in part over the same period as those on self-fertilization, namely
during the spring and summer of 1913. The whole of the work was
done at the Zoological Station, Naples, and to the staff of this institution
I owe my very best thanks for the willing advice and help given to me
throughout my stay.

The fact of self-sterility in Ciona, whether comparative or absolute,
suggested that some substance may be exuded by the eggs which
paralyzes the spermatozoa of the same individual, but does not affect
those of other individuals — or there might be something within the
egg envelope which acts in the same way. To test this hypothesis
experiments were commenced by trying the action of suspensions of
crushed eggs in sea-water on the spennatozoa. It was soon found that
there was a very large number of questions with regard to the action of
such egg-extracts on spermatozoa used to effect ordinary cross-fertiliza-
tion which must be investigated and cleared up before the more special
question of the mechanism of self-sterility could be attacked. The
outcome of these investigations with cross-fertilization is recorded in
this part of the paper.

The results obtained are all based on numerical data. They
depend on the comparison of the proportions of eggs fertilized by sperm-

H. M. FucHS 261

suspensions after either the eggs or the sperm have been subjected to
certain preliminary treatments. Thus the validity of the results depends
altogether on the way in which the experiments were carried out ; that
is to say, on whether the conditions of experiment allowed of any such
numerical comparison of the after-effects and of deductions to be drawn
from them. For a detailed description of the precautions taken to
ensure that the experiments should be of an exact nature the reader is
referred to the Part on " The conditions of self-fertilization in Giona
intestinalis " since the methods used were identical with those employed
in this case. A short resum^ of the technique is, however, given in the
following section. It is thought that these methods will break a new
road for the investigation of fertilization phenomena, for work hitherto
done on this subject, in which the results have been based on numerical
counts, seems to have been carried out under such comparatively inexact
conditions that it is usually extremely difficult to judge the extent to
which the results are valid.

II. Methods.

The method of experimentation was the same as that employed in
investigating the conditions of self-fertilization in Ciona intestinalis. For
a detailed account of these methods reference should be made to Part I.

Essentially the method consisted in an exact comparison of the per-
centages of eggs fertilized by sperm-suspensions of identical concen-
trations, after either the eggs or the sperm had been treated in
different ways prior to the fertilization. The results of such treatment
were judged by the relative ease or difficulty with which fertilization
could subsequently be brought about. The sperm-suspensions used
were always so dilute that they would not cause the fertilization of all of
the eggs to which they were added. In this way comparisons could be
instituted, since the percentages of eggs fertilized in the different lots
lay between 0 and 100.

The term " fertilizing power " of a sperm-suspension is employed in
the following sense. If a sperm-suspension of a certain concentration
can bring about the fertilization of say 40°/„ of the eggs after it has been
treated in a definite way, whereas another suspension of the same
sperm, of identical concentration but not having been subjected to the
treatment, only causes 20°/^ of the eggs to be fertilized, it is said that
the "fertilizing power" of the sperm has been increased by this
treatment.

2*82 Studies in the Physioloffj/ of Fertilization

A capital letter (e.g. ^) is used to denote the eggs of a certain
female, and a small letter (e.g. 6) the sperm of a certain male. An
experiment to test the effect of solution X on the fertilizing power of
sperm b would be carried out as follow.s. The object is to inseminate
approximately equal quantities of A eggs with (1) a plain dilute
suspension of b sperm, and (2) a suspension of b sperm of identical
concentration as suspension (1), but to which solution X has been
added. To obtain (1) and (2), one sperm-suspension is made up and
diluted to the required amount by the further addition of sea-water.
Equal quantities are pipetted into two dishes. To the first dish is
added a certain quantity (n cc.) of sea- water; to the second an equal
quantity (n cc.) of solution X. These two suspensions (1) and (2) are
then added to the two lots of eggs and the effect of solution X is
judged by the relative numbers of eggs fertilized in the two dishes.

It is obvious that the whole validity of this method depends on the
degi-ee of exactness with which all other conditions are kept identical
for the two fertilizations, while the one fiictor alone — here the presence
or absence of solution X in the sperm-suspension — is altered. Of especial
importance is the identical concentration of the two (or more) sperm-
suspensions, and the complete mixing of the eggs and spermatozoa in
making the different fertilizations, so that all eggs have an equal chance
of being fertilized. The precautions taken to ensure this exactnessi
that is to say, the method of making up the sperm-suspensions, of sub-
dividing and treating the eggs and sperm, the way in which insemination
is effected, together with the method of counting the proportions of
fertilized and unfertilized eggs, are given in full in the Part on the
" Conditions of self-fertilization in Ciona."

The results of each series of experiments are tabulated, the numbers
in the Tables being the percentages of eggs fertilized, unless otherwise
stated.

Eggs from a single individual and sperm from another single
individual were used in each experiment. It goes without saying that
the presence of genital products of any other individual was always
rigidly excluded. All glassware and instruments were sterilized with
hot water before each operation, and the hands were dipped into hot
water. All sea-water employed was taken from the circulation and
passed through a Berkefeld filter. The method employed in removing
the eggs and sperm from Ciona intestinalis has already been described
in the part (Part I) on self-fertilization in this form. Similar methods
were used in taking the genital products from the other Ascidians used

H. M. FucHS 263

in the investigation, namely, Ascidia mentula and Phallusia mamillata.
Besides these forms, four species of Echinoids — Strong ylocentrotus
lividus, Sphaerechinus granuhiris, Echinus microtuherculatus, and Arhacia
pustulosa — were made use of, together with one Asteroid, Asterias
glacialis. The procedure in obtaining the eggs and sperm of the sea-
urchins was this. Each individual was washed under a stream of fresh-
water to destroy any adherent spermatozoa, after which it was opened
by an equatorial cut. The aboral half was then reversed over a dish of
sea-water, so that the genital pores just dipped beneath the surface. If
the animal was mature, large quantities of eggs or sperm as the case
might be were shed into the dish of water, with very little admixture of
body cavity fluid'. The eggs of Asterias were obtained by cutting open
the body wall, after the animal had been washed in fi-esh-water, and
removing portions of the ovary. These were placed in sea-water, which
was presently filtered through bolting silk to strain off the pieces of
ovarian tissue from the eggs.

The efficacy of these precautions was tested in every case by keeping
a sample of eggs separate, to which no sperm had been added. In no
experiment recorded here did a single egg in the control segment.

By " egg-extract " is meant a suspension of crushed eggs in sea-
water. The eggs were crushed between two sterilized glass plates and
the substance so obtained washed off the glass into sea-water. By this
means a veiy finely divided suspension can be made, and presumably
soluble substances present in the eggs go into solution in the sea-water.
The suspension was examined after having been made up, to be certain
that no uncrushed eggs were lying in it, the subsequent fertilization of
which would destroy the value of the percentages. Throughout the
work the egg-extract was made up of more or less the same concentra-
tion, although it is impossible to make up two samples of absolutely
identical strength. For every 12 cc. of suspension, two drops of eggs
were crushed between the plates and then added to the water.

" Ovary-extract " of Ciona was made up in the same way as the egg-
extract. In this case special precautions are necessary that none of the
spermatozoa from the testis have been removed with the ovary. To
check this possible source of error, unfertilized eggs from another
individual were in each case left lying in a sample of the ovary extract
and afterwards examined to see that none had segmented.

"Egg-water"isused to designate sea-water in which eggs have been
lying for a longer or shorter interval.

1 I am indebted to Dr Otto Koehler for bringing this method to my notice.

264 Studies in the Physiology of Fertilization

Giona blood was removed from the body in the following way. An
animal was washed in a stream of fresh-water. After this an incision
was made in the body-wall in the region of the heart, which then bulged
out through the cut. The animal was held over a small glass dish with
the cut downwards, and the projecting heart was then punctured, so
that the contained fluid flowed out into the dish. The comparatively
small volume of blood obtainable from one animal was usually in-
sufficient for carrying out an experiment, and in such cases the volume
of liquid was increased by the addition of sea-water. The possible
presence of spermatozoa in the blood was checked in the same way as
with the ovary-extract. Unfertilized eggs from another individual were
placed in a small quantity of the blood and later on examined to make
sure that none had segmented.

III. Effects of Egg- and Ovary-Extracts on Fertilization

Percentages.

1. Cross-fertilization^ of Ciona in egg-extract.

The first experiments were made to try whether, when cross-fertili-
zation of Giona was brought about in the presence of crushed eggs of the
same species, the percentage of eggs fertilized was raised or lowered.
As in all subsequent experiments the method was strictly comparative.
In each experiment, approximately equal numbers of eggs of one
individual were placed in equal quantities of (1) sea- water, and (2)
extract of the eggs of another individual. After a certain interval equal
quantities of well-mixed dilute sperm-suspension from a third individual
were added simultaneously to the two lots of eggs and mixed. When
in the 4-cell stage, the percentages of eggs fertilized in each lot were

counted ^

TABLE I.

(The bracketed numbers give the time in minutes after fertilization at which the
first 4-eell division was completed.)

In each experiment the eggs lay in (1) water, (2) extract for forty minutes before fertili-
zation.

Water Egg-extr&ct

37 (87) 100 (87)

12 (89) 95 (89)

3 ly

is here used to mean the fertilization of the eggs
of a hermaphrodite animal by the spermatozoa of another individual. No self-fertili-
zations were made in this investigation.

2 As in all the experiments described in this paper, samples of the lots of eggs were
kept unfertilized as controls, to guard against accidental contamination with foreign
sperm. This is to be taken as granted in the descriptions of subsequent experiments.

Exp.

I.

(IG.27.1.)

Exp.

II.

(16.28.1.)

Exp.

III.

(16.29.1.)

1 The term "cross-

fertilization

H. M. FucHS 265

The Table shows that in each of the experiments more eggs were
fertilized in the water containing the egg-extract than in plain sea-
water. The suggestion at once arose — were the eggs in the egg-extract
developing in part parthenogenetically { This was ruled out of court
by controls of unfertilized eggs in egg-extract, none of which segmented-

The egg-extract present at fertilization and during the early stage
seems to have no baneful effect on development. Table I shows that in
Exps. I and II the eggs in plain water completed the 4-cell division
simultaneously with those in the egg-extract. Moreover, in a further
experiment (17.29.1) in which the eggs B were fertilized by sperm a in
(1) water, (2) B egg-extract, (3) A egg-extract, (4) C egg-extract, the
resulting larvae all developed equally well.

Evidently some substance extracted from the eggs by breaking them
up in sea-water so affects the eggs or the sperm or both that more eggs
are fertilized than is the case in plain sea-water, when other conditions
are equal.

An experiment (22.7.2) was then made, in which equal quantities of
A eggs were put simultaneously into (1) fouV dishes, each containing
12 cc. sea-water, (2) four dishes, each containing 12 cc. B egg-extract.
At given successive intervals one drop of b sperm was added, at the
same moment, to (1) a dish of water containing A eggs and (2) a dish of
B egg-extract containing A eggs. Each was then well mixed.

TABLE II.

(The numbers in brackets give the time in minutes after fertilization at which the
first 4-celI division was completed.)

Eggs tried out into dishes containing (1) water, (2) extract at 12.20.

(1) Water (2) Extract

Cross: A/b, B extract. Fertilized 12.20 62 (88) 91 (88)

Fertilized 12.40 53 (89) 57 (89)

Fertilized 10 31 (89) 57 (89)

Fertilized 1.20 24 (84) 52 (84)

This experiment was, in the first place, a confirmation of the previous
ones, in that the eggs fertilized in the egg-extract all showed higher
fertilization percentages than those fertilized after a corresponding
length of time in water. In this case the egg-extract was obtained from
the same individual from which the sperm was used.

The Table also shows that although the fertilization percentages
decreased with each fertilization, the length of time taken to attain the
4-cell stage became shorter in the last fertilization (see Part I). As

266 Studies in the Phjisiology of Fertilization

in the last experiments, the segmentation rates were identical in each
pair of fertilizations for eggs in water and in egg-extract.

Finally the experiment shows that the extract has an immediate
effect in raising the percentage, since the first pair of fertilizations was
made as soon as the lots of eggs had been tried out into water and
extract. The falling off of the percentages in subsequent fertilizations
was less rapid in the eggs in extract than in those in water.

2. Cross-fertilization of C'iona in boiled egg-e.rtract.

An experiment was made (21.3.2) to discover whether heating the
egg-extract altered its effect on fertilization.

Extract was made of C eggs and a portion heated to boiling-point.
Approximately equal numbers of j4 eggs were placed in (1) 12 cc. water,
(2) 12 cc. unboiled egg-extract, (3) 12 cc. boiled egg-extract. After
33 minutes one drop of diluted sperm-suspension b was added to each.

TABLE III.

Water Extract Boiled extract
Cross: Ajb, C extract 19 8

The counts of eggs fertilized show that both boiled and unboiled
extracts raised the percentages to an almost equal extent. The difference
of 1°/^ between the two Ijes within the limits of experimental error.

Thus the value of the egg-extract is not destroyed by boiling for a
short time. No further experiments were made on the stability of the
extract as there were more imjjortant questions to be decided first.

3. Cross-fertilization of Ascidia in egg-extract.

The question then arose as to whether the effect of egg-extract on
fertilization percentages was confined to Ciona or was to be found in
other forms as well. The next animal to be tried was Ascidia mentula.

Approximately equal numbers of eggs D were placed in each of
(1) two dishes containing 17 cc. of sea- water, (2) two dishes containing
17 cc. C egg-extract. Thirty-three minutes afterwards, two drops of a
weak sperm-suspension c were added (1) to a dish of water containing
eggs, (2) to a dish of egg-extract containing eggs. At the same time
five drops of a stronger sperm-suspension c were added (1) to a tlish of
water containing eggs, (2) to a dish of egg-extract containing eggs.
The experiment was thus double, the comparison being made first with
weaker and then with stronger sperm.

H. M. FucHS 267

TABLE IV. (1.2.2.)

(I) Water (2) Extract
Cross : D/c, C extract Weaker sperm 0 8

Stronger sperm < 1 36

In both parts of the experiment the fertilization percentage was
greater in egg-extract than in water. It will be shown later on that
this is not only the case for Ciona and Ascidia, but is the same for all
the forms tried.

4. Gross-ferlilization of Ciona in ovary-extract.

In the following experiment, a comparison was made between the
percentage of eggs fertilized (1) in plain sea- water and (2) in ovary-
extract, instead of egg-extract.

Approximately equal amounts of A eggs were placed in (1) 12 cc.
water, and (2) 1^ cc. C ovary-extract. After 38 minutes each was
fertilized by the addition of one drop of b sperm-suspension. Besides
the usual control of unfertilized eggs A, some of the latter were also put
into C ovary-extract to guard against the accidental presence in this of
sperm from the testis of the same animal'.

TABLE V. (20.L2,)

(1) Water (2) Ovaiy-extraet

Cross:_ Ajh, C extract ... 86 98

As shown by Table V, the presence of the ovary-extract caused an
increase in the percentage of eggs fertilized.

The next experiment was made on similar lines, but three different
concentrations of ovary-extract were used.

Approximately equal amounts of eggs E were tried out into four
dishes containing respectively equal quantities of water and of three
different concentrations of D ovary-extract, as is shown in Table VI.
After 34 minutes, each was fertilized by the addition of one drop of _/
sperm-suspension.

TABLE VL (20. L2.)

15 cc. water S cc. water

16 cc. water 1 cc, extract 8 cc, extract 16 cc. extract

Cross: £//, D extract 68 91 92 94

As is seen from the above Table, this experiment was a confirmation
of the last. (The weakest extract was here almost sufficient to raise
the percentage of fertilization to the maximum.)

' This was done in all other experiments in which o\ary-extract was used.

268 Studies in the Phijsiology of Fertilization

5. Gomparison of the effects of egg- and ovary-extracts on the
fertilization in Ciona.

The increase in the number of eggs fertilized in the presence of the
extracts might be due to some substance contained in ripe eggs alone.
In the case of the ovary-extract, it would then be derived from ripe
eggs present in the lumen of the ovar}'. Whether this is so, or whether
the substance is contained in the ovarian tissue itself can be determined
by making a comparison of the effects of equal concentrations of egg-
and ovary-extracts on the fertilization. The following experiment was
made to determine this point.

Approximately equal numbers of B eggs were placed in 16 cc. of
(1) water, (2) A egg-extract in three concentrations, (3) A ovary-extract
in three corresponding concentrations. The strongest egg- and ovary-
extracts were made up in approximately equal conceritrations by taking
as nearly as possible equal volumes of eggs and ovary, and washing the
juices into equal quantities of water. Fertilization was effected by
the addition of one drop of c sperm-suspension to each of the dishes.

TABLE VII. (20.4.2.)

16 cc. water IS ec. water 8 cc, water 0 cc. water

0 cc. extract 1 cc. extract 8 cc. extract 16 cc. extract
Cross : ll/c, A extract

Egg-extract...) ^ (2 13 33

Ovary-extract ( (3 21 36

The Table shows that an increasing number of eggs were fertilized
in increasing concentrations of both egg- and ovary- extracts. The
numbers were, however, not identical in corresponding concentrations
of the two extracts, so that the presence of the substance which aids
fertilization in equal amounts in both ripe eggs and ovary is not proved.
Indeed the ovarian extract had a greater effect than that derived from
the eggs. It must be remembered, however, that it is exceedingly
difficult to obtain egg- and ovary-extracts in exactly equal concentra-
tions, and to such a difference in concentration the differences in effect
shown in the Table might easily be due.

6. Cumparison of the effects of "foreign" and "own" egg-extracts
on the cross-fertilization percentages in Giona^.

The experiments with egg-extracts were originally made to test
whether there is some substance in the eggs of Giona which hinders

' "Foreign" means from another, and "own " from the same individual.

H. M. FucHS 269

fertilization by their " own " sperm, i.e. by sperm from the same indivi-
dual. It was thought that if there were such a substance, its presence
in the sea-water should prevent sperm taken from the same individual
as itself from cross-fertilizing the eggs of another individual. That
such is not the case is demonstrated by the experiments shown in
Tables II and IV, in which the extracts were taken from the same
individuals as those from which the sperm was derived. These extracts
caused an increase in the number of eggs fertilized in their presence.

It might be, however, that an egg-extract from the same individual
as the sperm had a lesser stimulating effect than one from a " foreign "
animal. In the much smaller concentration in which the substance
must be present at the surface of eggs in the sea than in artificial egg-
extracts, "own" extract might have no perceptible favouring influence
on fertilization, while the influence of that from another individual
would be great enough to be effective. In this way, the difficulty of
effecting self-fertilization could be accounted for.

The h3rpothesis was put to the test in the following experiment.
Approximately equal amounts of ^ eggs were put into (1) 10 cc. water,

(2) 10 cc. of egg-extract derived from the same animal as the eggs (A),

(3) 10 cc. of egg-extract derived from the same animal as the sperm (B),
and (4) 10 cc. of extract derived from a " foreign " animal (C). After
35 minutes, one drop of b sperm-suspension was added to each dish.
It should be noted that the three extracts were made as nearly as
possible of equal concentration.

TABLE VIII. (17.29.1.)

Water A extract B extract

C extract

Cross :

Ajb ... 21 63 62

63

The Table shows that the extracts derived from the three indivi-
duals had practically the same stimulating effect on fertilization. The
difference of 17o li^s within the limits of experimental error.-

The foregoing experiments show that self-sterility in Ciona cannot
be accounted for by the presence of a substance in the eggs of an
individual B, which not only prevents the union of B eggs with
b sperm, but also of "foreign" eggs (A) with b sperm. Nor is it due
to something contained in B eggs which favours fertilization of eggs A
by b sperm relatively less than does a substance contained in the
" foreign " eggs. At any rate, if such an inhibiting substance be
present, its effect is masked by stimulating substances contained in
the egg-extracts.

270 Studies in the Physiology of Fertilization

The most important experiments in regard to this question, however,
are those to find the influence of egg-extracts, both from " own " and
'' foreign " eggs on self-fertilization itself Some such experiments have
been made already, but these must be extended and confirmed before
being published.

IV. Does the Extract affect the Egos or the Sperm ?

The experiments already described have shown that if a given
number of eggs be mixed with a certain concentration of sperm-
suspension in sea-water, fewer eggs segment than if approximately
the same number of eggs are inseminated by exactly the same con-
centration of sperm in egg- or ovary-extract, other factors remaining
the same. Now this result might be due to a " stimulating " effect
of the extract on the spermatozoa alone, or to some change brought
about in the eggs so that they can be more easily fertilized, or to a
combination of both causes. The experiments described in the following
sections were made to settle this question.

1. Effect of extract on the sperm of Ciona.

In each of the experiments recorded in Table IX, a sperm-suspension
was made up in the usual way. 5 cc. of this was mixed with 5 cc. of
water (= Sperm a), and at the same time 5 cc. was mixed with 5 cc.

TABI.E IX. (23.10.2.)

a=5 cc. sperm-susp<

^ = .5 cc. sperm-suspeusion + .5 cc. extract.

„ . , . ^ I a= 5 cc. sperm-suspension + 5 cc. water,

Sperm in each experiment : <

(1)

a sperm

(2) n spbnn + l drop

extract added to water

at fertilization

(3) ^ sperm

Exp.

1.

£//.

D extract ...

<1

<!

6

Exp.

2.

Hji,

G extract ...

0

0

14

Exp.

3.

Jjl,

J extract ...

0

0

17

Exp.

4.

Njo,

il extract ...

0

0

11

Exp.

5.

W/9.

M extract ...

5

5

12

of the egg-extract (= Sperm /3), suspensions a and ^ being thus of
identical concentrations. The eggs to be fertilized were divided into
three equal lots, each in 16 cc. sea-water. Thirty minutes after the sperm
mixtures had been made up, fertilization of the three lots of eggs was
effected as follows : — (1) addition of one drop of Spi'i'ni a ; (2) addition
of one drop of Sperm a, and at the same time one drop of egg-extract;
(3) addition of one drop of Sperm /3. The fertilization (2) was made to

H. M. FucHS 271

test whether or not the drop of egg-extract necessarily introduced with
Sperm /S itself influences the fei-tilization, or whether Sperm /8 had
been altered by its treatment with extract.

The experiments all agreed in showing that if two suspensions of
sperm from a given animal are made up of equal concentrations in
(a) water and (b) egg-extract, suspension (6) is capable of fertilizing
more eggs than is (a). The addition of a drop of extract to the eggs
at the same time as the sperm from the water-suspension (a) caused no
increase in the percentage of eggs fertilized (see column (2) of Table),
proving that the presence of this small quantity of extract at the
moment of fertilization was not reason for more eggs being fertilized
by (h). Evidently the sperm which had been treated with egg-extract
before being added to the eggs was stimulated in such a way that more
spermatozoa were capable of fertilizing eggs than was the case when
the sperm had previously been in sea- water alone.

Two more experiments were made confirming the above conclusions.
In each case the sperm was treated, before being used to fertilize the
eggs, with different concentrations of egg-extract. Moreover, instead
of lying in the extract for half an hour before being used, as was the
case in the last experiments, the suspensions were added to the eggs
as soon as they had been well mixed by pouring. The results showed
that in the short time necessary for the latter operation, the sperm was
already stimulated. Increasing numbers of eggs were fertilized by
sperm from increasing strengths of extract.

The details of the experiments are as follows :

In Exp. 1 the sperm-suspension was made up and then three drops
were added to each of four dishes containing (1) 10 cc. sea-water, (2) 8 cc.
water -|- 2 cc. extract, (3) 4 cc. water -l- 6 cc. extract, (4) 10 cc. extract.
These four liquids, containing identical concentrations of sperm, were
poured on to four equal quantities of eggs in separate dishes.

In Exp. 2, three drops of sperm-suspension were added to (1) 10 cc.
water, (2) 7 cc. water-|-3 cc. extract, (3) 10 cc. extract. These were
then poured on to three equal quantities of eggs.

Table X gives the result of the experiments, which confirm those
of Table IX, and also show the effects of varying concentrations of
extract on the sperm.

TABLE X. (34.16.4 and 34.21.4.)

Sperm (1)

Sperm (2)

Sperm (3)

Sperm (4)

Exp. 1.

A/b, C extract

8

12

74

82

Exp. 2.

D/c, F extract

<1

52

09

—

272 Studies in the Phxjaiohujtj of Fertilization

2. The effect of extract on the eggs of Giona.

The experiments described in the preceding section showed clearly
that treatment with egg-extract increases the fertilizing power of a
sperm-suspension. That is to say, such a suspension is capable of
fertilizing more eggs than sperm of equal concentration made up in
plain sea-water, other conditions remaining unchanged. This does not
prove, however, that the increased percentages of eggs which segmented
in the initial experiments when fertilized in the presence of extract,
over those fertilized in plain water with the same amount of sperm,
were to be attributed only to the action of the extract on the sperma-
tozoa. The substances in the egg-juice might also have influenced the
eggs, making them more receptive to spermatozoa. In the experiment
to be described next, the action of the extract on the spermatozoa was
as far as possible eliminated. Eggs were treated with extract, and the
proportion subsequently fertilized by the addition of a given amount of
sperm was compared with that in eggs not previously so treated.

The eggs of an individual were divided into two lots and placed
in (1) water, and (2) ovary-extract, and left for 43 minutes. After
this interval, approximately equal numbers of eggs were tried out as
follows :

((') Eggs in 1 cc. water, from (1).

(6) Egg.s in 1 cc. extract, from (2).
(c) Eggs in 1 cc. extract, from (2).

Immediately before fertilization a sperm-suspension was made up,
and equal amounts were dropj^ed into («) 80 cc. water; {^) 30 cc. water;
(7) 30 cc. extract. Fertilizations were effected by pouring (a) on to (a) ;
(/3) on to (6) ; and (7) on to (c). The large amount of water (30 cc.)
added to the eggs with the sperm was to dilute the extract in (h) as
much as possible, in order to prevent action on the sperm.

The experiment was made double, the fir.st series being when
three drops of sperm were added to (a), (/S), and (7), and the second
when ten drops were added to each.

TABLE XI.

Bjc, A extract.

I

Eggs (a) from water
Sperm (a) from water

11
Eggs {b) from extract
Sperm (3) from water

III

Eggs ('•) from extract
^perm (v) from extract

3 drops sperm ..
10 drops sperm ...

1

5

2

52

H9

The comparison of the percentages of eggs fertilized in I and II in
the Table shows whether or not the extract had affected the eggs, which

H. M. FucHS -273

had lain in it befbi'e being fertilized. Ill was made to show the effect
of extract on the sperm, for comparison.

The Table shows that in both parts of the experiment — with the
stronger sperm and with the weaker sperm — the pei'centages in II were
1 °/„ higher than those in I. This fact may be due to one of three
causes. It may be experimental error ; but the fact that the difference
is the same in both parts of the experiment argues against this. Again,
it may be that the eggs (b) were affected by their stay in the extract
previous to fertilization, so that they became more easily fertilizable —
although to a very slight extent. Lastly, the increase may have been
due to the action of the 1 cc. of extract introduced with the eggs (b)
on the sperm, at the moment of fertilization. A comparison of these
figures with those in III, which shows the very large increase in the
fertilizing power of the sperm caused by a previous treatment with
extract of the full strength, shows that the last of the three possible
explanations given above is almost certainly the correct one.

Thus from the experiments recorded in Tables IX, X and XI, we
can draw the conclusion that the excess in the number of eggs fertilized
by a given amount of sperm in the presence of extract over the number
fertilized in jjlain water is due to an action of the extract on the sperm
and not on the eggs.

3. Effect of egg-extract on the fertilizing pmoer nf the upervf of
Strongylocentrotus.

The experiments already recorded on the influence of egg- and
ovary-extracts on the proportion of eggs fertilized by a given con-
centration of sperm-suspension have, with one exception, all been
made with Ciona intestinalis. In the following section, experiments
are described on the effect of egg-extract on the fertilizing power of
the spermatozoa of Strongylocentrotus liviclus.

The first experiment is a plain comparison of the effects of fertilizing
with sperm-suspensions of equal concentration, made up in (1) plain
sea- water, and (2) egg-extract. The experiment was made for four
different strengths of sperm.

A sperm-suspension was made up, and a given number of drops,
added to (1) 10 cc. sea-water, (2) 10 cc. extract. Each of these were
then poured on to approximately equal numbers of eggs.

The Table shows that the extract had the same effect on the per-
centages of eggs fertilized as was the case with Ciona and Ascidia.

If equal amounts of the same sperm-suspension be added to equal

Jouni, of Gen, jv 18

Water

Extra

^ 1 drop spei'm
4 drops sperm ...

0

7

<1

42

8 drops sperm ...
12 drops sperm

<:1

49

1

Hfi

274 Sfmlics ill lli( rinisliihiiiji <if Fertilization

quantities ol' (1) plain sua-water and (2) egg-extract, and a <lit))) of
each compared under the microscope, the spermatozoa are very much
more active in the extract suspension. Moreover, if eggs be ])ut in

TABLE XII. (1.26.1)

Cross : A jc A egg-extract

each of the ih-ops, the number of spermatozoa which collect round the
eggs is considerably greater in the latter suspension. These effects,
which are very obvious in Strongylocentrotus, are hardly noticeable in
Ciona, where the sperm is always much more sluggish in its movements.

The greater activity of the spermatozoa of Stroiigylocentrotus in the
presence of egg-extract suggests that the greater proportion of eggs
fertilized by such suspension is due to UKjre spermatozoa coming into
contact with eggs, owing to their quicker movement. This is, however,
at all events not the whole explanation, since Lillie (3) has shown that
the egg-extracts have a positive chemotactic effect on the spei-matozoa.
as well as an activating influence.

The following two experiments were made to determine the effect
on the fertilizing power of the sperm of treatment for varying lengths
of time with egg-extract.

In each experiment a sjjerm-suspension was made in sea-water and
1 cc. was pipetted into each of (I) four dishes, each containing 5 cc. of
plain sea-water, and (2) four dishes, each containing 5 cc. of egg-extract.
These suspensions of equal concentration were used for fertilization
immediately and at ten minute intervals. At each fertilization, 5cc.
plain water was added to (1) a dish of water sperm-suspension, and
(2) a dish of extract sperm-suspension. After the usual thorough

Exp. 1. Ajb

Exp. 2. Ajr

TABLE XIIT.

(4.7.0.)

Times of fertilization
after making sperm-
suspensions (1) and (2)

(1) Water

(2) Extract

' Immediately . , .

(jl

G9

10 minutes

10

53

20 ..

4

27

30

0

8

Immediately ...

89

93

10 minutes

17

72

20

4

32

30

<1

10

H. M. FucHS 275

mixing, these were poured separately on to approximately equal amounts
of eggs.

In the two experiments the fertilizing power of the sperm-suspension
decreased rather rapidly, both in water and extract, but at each suc-
ceeding fertilization more eggs were fertilized by the extract-sperm than
by the water-sperm. These experiments are a parallel to that recorded
in Table II for Ciona.

F. R. Lillie (15, p. 558) states that " the powerful effect of the egg-
extract on spermatozoa of the same species may be shown by a complete
loss of motility, as we have already seen, and also by a cori'esponding
loss or diminution of the fertilizing power." He then describes an
experiment illustrating this. To five watch glasses containing each
eight drops of water oi- of different concentrations of egg-extract were
added thiee drops of " opalescent" sperm-suspension. After 12 minutes
" a th'op of a suspension of fresh eggs was added to each." 5 "/„ of the
eggs in the water segmented, but none of those in the four different
concentrations of extract.

That this was quite contrary to my own results is clearly seen in
the experiments recorded in Table XIII. I determined, however, to
repeat exactly the experiment of Lillie's described above. I made the
fertilizations in the very small amounts of liquid which Lillie used,
although my preliminary trials had shown that this is an inaccurate
method, as, under these conditions, the eggs cannot be rapidlj' and
uniformly mixed in the different vessels.

The details of the experiment are as follows. To each of four watch
glasses, containing respectively eight drops of water and eight drops of
difi'erent concentrations of extract as given in Table XIV, were added
three drops of a sperm-suspension. 12 minutes later, one drop of eggs
was added to each watch glass. Tw<i such experiments were made.

TABLE XIV. (4.24.6.)

5 drops water 3 drops water
8 drops water 3 drops extract 5 drops extract 8 drops extract

Exp. 1. J/c, A extract 0 <! 4 8

Exp. 2. Ajd, B extract -5 13 35 .57

Thus, in both experiments, treatment for 12 minutes in all three con-
centrations of egg-extract increased the fertilizing power of the sperm.
The most important points in which these two experiments differed
from that of Lillie quoted above, are the following: (1) Lillie's experi-
ments were made with Arhacia, mine with Strongylocentrotus; (2) Lillie's
extract was probably stronger: (3) I u.sed a weaker suspension of sperm.

18—2

*J7() SfHflu's III the /*/ii/siolof/i/ of FertUization

Another experiiaeut was then made, on exactly the same lines as
the last, but in which the extract was made up of eight times the usual
strength. This experiment was made double, two different strengths
of sperm being used to each concentration of extract. The result was
exactly parallel to those of the preceding experiments. It is given in
Table Xy.

TABLE XV. (+.24.6.)

S (Irojis water

Silrops water
h drops extract

8 drops ext

rapt

0

B2

35

;s

(iH

92

, I Wealier sperm

Bid, B extract , _,

I Stronser sperm.

Finally the experiment was repeated with thick sperm. As was to
be expected, 100 "/ fertilized in water as in all the concentrations of
extract, but the sperm-halos round the eggs were progi-essively larger
in increasing concentrations of extract.

Thus I cannot agree with Lillie's conclusion that the fertilizing
power of sperm which has stood in egg-extract is decreased. On the
contrary, the experiments just described, together with others recorded
in this paper, show clearly that treatment with egg-extract not only
has an immediate effect in increasing the fertilizing power of the sperm,
but suspensions in the presence of extract keep the power of being able
to fertilize more eggs than suspensions of the same sperm of the same
concentration in plain sea-water.

V. Effects of Egg-waters on the Fertilizinc; Power of

THE Sperm.

The experiments described up to now have shown that a substance
or substances can be artificially liberated from the eggs — by breaking
them up in sea-water — which stimulates spermatozoa. It remains to
be seen whether or not such a substance is noi-nially secieted by the
eggs into the sea-water. If not, we have been dealing with a purely
artificial phenomenon, but if there is a normal secretion of a sperm-
stimulant by the eggs, its inv(>stigation must be of the highest im-
portance for the understanding nf the fertilization process. In the
experiments recorded below, eggs were allowed to remain in a relatively
small amount of water for some time, and this water was then used in
the same way as the extract had been in previous experiments. Such
sea-water, in which eggs have been allowed tn stand, I call "egg- water"
for the sake of brevity.

H. M. FiTCHs 277

1. Experiments luith Cioiiu intestinalis.

In Exps. 1 and 2 (Table XVI), eggs from three individuals' were
kept in 3 — 4 times their volume oi water for 5} hours, to obtain the
" egg-water." Given amounts of sperm-suspension were dropped into

(1) 10 drops of plain water, (2) 10 drops of the egg- water. 10 cc. water
was then added to (1) and (2), and they were poured on to appi-oximately
equal amounts of eggs from a given individual.

In Exp. 3, eggs from several individuals lay in 3 — 4 times their
volume of water for two hours. The rest of the treatment was the same
as in Exps. 1 and 2.

For Exp. 4 an individual was found with an exceptionally large
number of eggs. Some of these were allowed to stand in 10 times
their own volume of water for two hours to obtain the egg- water. Given
amounts of sperm-suspension were dropped into (1) 1 cc. of water,

(2) 1 cc. of egg-water. (1) and (2) were then poured on to equal
amounts of eggs from the same individual from which the egg-water
had been obtained.

The results of the experiments are given in Table XVI.

TABLE XVI.

Plain water Egg-water

n , , „ »r- 1 . I 3 drops sperm ... 2 11

Exp. 1. Alb, Mixed egg-water -' „ ,„

( o ,, ,,-,.. o io

TH 1 ^;j «»• 3 i I 2 di'ops sperm ... 0 0

Exp. 2. C/rf, Mixed egg-water • ,

T-. o r, 1 ■ .»•• -1 X I 2 drops sperm ... 2 5

Exp. 3. EU, Mixed egg-water -j ^ ^ ^ ^.^ ^^

Exp. 4. Glk, G egg-water ... ^^ ^'"J' ^'-"^ ' • ^J ^J

These experiments show that for Ciona, water in which eggs have
lain has the same stimulating effect on spermatozoa that artificial
egg-extracts have.

2. Experiment with Arbacia pustulosa.

This experiment was made on exactly the same lines as those just
described with Ciona. Egg-water experiments are, however, much more
easily made with Echinodejms than with Ciona, since a much greater
volume of eggs can be obtained, and hence more water containing the
egg-secretions for experimentation.

' I need hardly repeat that as in all other experiments recorded in this paper, controls
were kept. Unfertilized samples of all the eggs used for egg-waters were put on one side
and afterwards examined to see that none had segmented.

278 Studies in tlie Phtjuioloyi/ of Fertilization

Eggs A were washed twice by allowing them to settle in finger-
bowls full of watt'r. They were then placed in a tube in 3 — 4 times
their volume of water. After 35 minutes, this water was drawn off
and a .second lot added to the eggs. When the latter had been in this
water for one hour, it was drawn off and used for the experiment.
Given amounts of sperm-suspension c (.see Table XVII) were added
to (1) 2 cc. plain sea-water, and (2) 2 cc. egg-watei-. To each of these
10 cc. of plain sea- water was added, and they \\i-\e poured on to separate
equal lots of eggs B.

TABLE XVll. (1.2.5 )

I'lain water Kgg-wat«r

„, , X t ^ drops sperm ... 35 89

Blc ^egg.water ^ j,, ^^ ^ ^^ g^ ,,,j

Thus Arbacia eggs behave as Ciona in secreting a substance into
the water, which increases the fertilizing power of the sperm of the
.same species.

3. Experiments with Strongylocentrotus lividus.

The four experiments given in Table XVIII were all made with the
same egg-water. Eggs were taken from three females and washed twice
to remove all coelomic fluid and ovarian tissue, by allowing them to
settle in finger-bowls full of water. They were then put in about five
times their volume of water, which was drawn off at varying intervals
(given in the Table) to be used for the experiments.

Just before the fertilizations were made, given amounts of sperm-
suspension were added to (1) 2 cc. plain water, and (2) 2 ec. egg-water.
After 1 — 2 minutes 10 cc. plain water was added to each, and they were
then poured on to approximately equal lots of eggs.

TABLE XVIII. (3.14.5.)

Plain water Kgg-water

„ , ,„ ,, . rr ■ , 1 3 drops sijerm 48 7H

Exp. 1. Ah, t-gg-water 55 miuutes on eggs ' „„ ,„„

I 1 cc. sperm 97 100

„.,,,,, L ^r- ■ L 1-5 drops sperm 45 58

Exp. 2. Ale Lgg. water 65 m.nutes on eggs -j ^ ^^ ^^ J^ ^^ ^^^

„ ., ,, n ■ ,„/^ • . 1 3 drops speriii 18 30

Exp. 3. A e. Egg-water 100 minutes on eggs '

' ' • be *'^ I 1 cc. sperm ... 5!» 84

]-, . , ,^. T^ .,,<>■ i I 3 drops sperm 8 17

Exp. 4. Ah, Egg-water I'iO minutes on ego-s-i ,

I 1 cc. sperm . . 3.j .53

Table XVIII shows that the eggs of Strongylocentrotus give off a
substance, or substances, into the sea-water which stimulates the sperm
of the species in the same way as we saw the case for Civna and
Arhacia.

H. M. FucHs 270

VI. Effect of Ciona Blood on the Fertilizing Power of

Cjona Sperm.

Trials were next made to see whether there is an agent which
increases the fertilizing power of the sperm in the blood, as well as in
the eggs of Giona. Owing to lack of time no other tissues of the body
were tested, but it will be important to do so in the future.

The blood was removed from the body by the method already given
(see p. 264). In each experiment the blood of one particular individual
(given in the Table) was used. After removal from the body it was
diluted to varying extents, according to the amount obtained. Given
amounts of sperm -suspension were added to (1) 2i cc. water, and
(2) 2i cc. blood. T(i each was added 10 cc. water, after which (1) and (2)
were poured on to approximately equal numbers of eggs. Each experi-
ment was made double, two different amounts of sperm being used.
The results of the experiments are given in Table XIX.

The Table shows that this series is an extensive one, there being
22 double experiments, or 44 comparisons in all. The reason for this
is not that this investigation is more important than previous ones, but
that, as will readily be seen from the Table, the results of each of the
Series A, B, C, D and E contradict one another, although the experi-
ments within each series agree. For the experiments in Series A and D
show an increase in the number of eggs fertilized as a result of the
treatment of the sperm with blood, while in Series B, C and E the per-
centages are decreased. The only exceptions to this statement are that
in Series D, all other experiments of which showed an increase in the
percentages as the result of the blood treatment, the second parts of
Exps. 10 and 15 gave no change'. Owing to the contradictory results
of the different series, the experiments were continued until a reason
for this behaviour was found.

At first it was thought that an explanation of the different effects of
the blood on the sperm might be that the behaviour of the former
differed according as it was derived from the same animal as the eggs
used in the same experiment, or as the sperm, or from a third individual.
That this is not the explanation is seen from the fact that Series D, in
which the percentages were raised by treatment of the sperm with
blood, there are experiments in which the blood was derived from (1)

• The first parts of Experiments 9 and 13 allow of no comparisons, as too little sperm
was used. The same is the case with the second parts of Experiments 20 and '21, where
too much sperm was used.

•J8() Studies ill the PliijHiolotjy of Fertilization

TABLE XIX. (41.4.6 e< seq.)

Series A. Exp. 1.
Exp. 2.
Exp. 3.

Ajb, A blood
C/rf, C blood
Elf, F blood

3 drops sperm
1 cc. sperm
j 3 drops sperm
/ 1 cc. sperm
( 3 drops sperm
j 1 cc. sperm

Series B.. Exp. 4.
Exp. 5.
Exp. 6.
Exp. 7.

/(/i, B blood
C/d, D blood
A'/;, F blood
«/''- ^/ blood

( 3 drops sperm
/ 1 cc. sperm
J 3 drops sperm
I 1 cc. sperm
\ 3 drops sperm
( I cc. sperm
\ 3 drops sperm
I 1 cc. sperm

Series C. ^Exp. 8.
"- Exp. 9.

Ilk, K blood
Ljm, L blood

3 drops sperm
1 cc. sperm
3 drops sperm
1 ce. spenii

Series D. Exp. 10.
Exp. 11.
Exp. 12.
Exp. 13.
E.\p. 14.
^ Exp. 15.

Exp. 16.
Exp. 17.

Alb, C blood
Ajd, D blood
FIf, G blood
E/li, J blood
A/A-, ./ blood
M/ii, M blood
.l//i>, a blood
J//P, P blood

3 drops sperm
1 cc. sperm
S 3 drops sperm
( 1 cc. sperm
( 3 drops sperm
( 1 cc. sperm
j 3 drops sperm
\ 1 cc. sjierm
3 drops sperm
1 cc. sperm
\ 3 drops sperm
( 1 cc. sperm
\ 3 drops sperm
( 1 cc. sperm
I 3 drops sperm
( 1 cc. sperm

Series E. Exp. 18.
Exp. 19.
Exp. 20.
Exp. 21.
E.\p. 22.

Qjr, S blood
Il/q, S blood
V/(, T blood
I'/w, W blood
A/r, .V blood

( 3 drops sperm
( 1 cc. sperm
3 drops sperm
1 ce. sperm
i 3 drops sperm
( 1 cc. sperm
( 3 drops sperm
( 1 cc. sperm
{ 3 drops sperm
( 1 cc. sperm

Water

41

85

0

<1

0
0

39
89
46
84
44
96
14
22

16

62

<1

10

71

31

83

12

37

0

0

1

7

3

14

18

49

<;1

4

33

95

7

57

lOU

100

100

100

77
100

Hlood
74
88

<1

10

<1

6

5
7
23
68
27
75
5
10

<1

23

<1

6

27
70
65
89
26
48

0
2

21
27

8
14
33
74

8
19

<:1
5

5

9

93

100

94

100

51

89

H. M. FucHS 281

the same animal as the eggs, (2) the same as the sperm, and (3) a third
animal. Similarly in Series E, where the blood caused a decrease in the
fertilization percentages, all three types are present.

What must be the true explanation of the phenomenon is seen from
the following list giving the times after they were brought into the
Aquarium at which the animals were used for experimentation.

Series A. Animals brouglit in from the sea the same day as the experiments
were made.

Series B. Animals liad been in the laboratory two weeks.

Series C. The same lot of animals as Series A, but used the following day.

Series D. Animals brought in from the sea on the same day as they were used.

Series E. The remainder of the same lot of animals used for Series D, but experi-
mented with two days afterward.s.

For Series A and D, in both of which the blood caused an increase
in the percentages of eggs fertilized, the animals were brought in fresh
from the sea on the same day. For Series B, C, and E, on the contrary,
the animals used had been in the Aquarium circulation for lengths of
time varying fi-om one day to two weeks, and it was in these experiments
that the blood produced a decrease in the percentages.

Evidently, then, even one night in the Aquarium water caused some
change in the composition of the blood of Ciona, causing it to reverse its
effect on the fertilizing power of the spermatozoa. It can hardly be
that this change is the first stage in the death of the animal, since all
unhealthy looking individuals were discarded, and animals such as were
used for the experiments were capable of living in the Aquarium circu-
lation in apparently good condition, for weeks after they were brought
in. That physiological changes occur in these animals, however, is seen
not only in the different behaviour of the blood described above, but in
the progressive decrease in egg-production, and in the fact that the eggs
are less and less pigmented each day'.

For the experiments on egg-extracts and egg-waters of Ciona, which
have been described in the jjreceding sections, animals were used which
had been in the circulation for varjring lengths of time after they were
brought in from the sea, although most were used fresh the same day.
Results were, however, uniform, and the following experiment, made
with animals which had been five days in the Aquarium is further proof
that there is no change in the eggs as there is in the blood.

' The eggs of Ciona which has been reared in the Aquarium are always more or less
unpigmented.

Water

Egg-extract

1 3 drops sperm

<1

.3

1 1 cc. sperm

2

10

282 Studies in the Phjisiolorjii of Fertilization

Given amounts of sperm-suspension (see Table XX) were added to
(1)5 cc. water, and (2)5 cc. egg-extract. To each was then added 5 cc
of water and they were poured on to separate lots of eggs. The experi-
ment was made with two different amounts of sperm. Table XX gives

the result.

TABLE XX. (42.4.7.)

Cross: .///;

VII. Influence of Egg-waters and Egg-extracts of Other
Animals ox the Fertilizing Power of Cioxa Sperm.

The reason why eggs can, in general, only be fertilized by sperm of
the same species has always been a puzzle to physiologists. The
experiments on egg-secretions suggest that the action on the .spermato-
zoon of a substance given off by the egg is a necessary- preliminary to
fertilization. Now, if the secretions of the eggs only affected the sperm
of the same species, but had no action on the spei'm of other species,
this might be an explanation of the specificity of the fertilization process.
To te.st thi.s hypothe.si.s, the actions of the egg-extracts and egg-waters of
Phallusia, Arbacia and Strongylocentrotus on Giona sperm used to
cross-fertilize Giona eggs were tried.

1. Phallusia egg-extract.

This experiment was made before it had been settled that the
action of extracts was on the sperm and not on the eggs. In consequence
the eggs of Giona (A) were allowed to stand in five dishes, the first of
which contained 16 cc. of sea-water, and each of the others IG cc. of
different concentrations of Phallusia egg-extract, f<ir 'M^ minutes. After
this interval a drop of Giona sperm-suspension {b) was added to each of
the dishes. The result was as follows :

TABLE XXI. (21.6.2.)

Co. sea-water

Cc. Phallusia egg-extract

Cross: Cionii A/t>

2. Arbacia egg-extract.

In the next series of three experiments, the Giona sperm was treated
with Arbacia egg-extract before being used to cross-fertilize Giona eggs.

1()

15

10

-*)

0

0

1

l>

11

16

4

8

20

21

27

H. M. FucHS 283

Given aniaunts of Ciona sperm-suspension (set' Table XXII) were added
to (1) 3 cc. plain sea-water, and (2) 3 cc. Arbacia egg-extract. 10 cc.
water was then added to each, and they were poured on to approximately
equal amounts of Ciona eggs. Each experiment was double, two
different quantities of sperm being used.

From Tables XXI and XXII it is seen that percentages of eggs
fertilized in Ciona by a certain concentration of sperm is raised by
treatment of the sperm with Phallusia or Arbacia extract, in the same
way as it was by treatment with extract of the eggs of the same species.

TABLE XXII. (38.6..5.)
( 3 drops sperm

Exp. 1. Ajh ^ ^2

Exp. -1. C/d j.^

3 drops sperm
2

( 3 drops sperm

k'ater

Arhuria
egg-extract

76

88

90

97

2

15

25

46

0

2

<^1

10

Exp. 3. t'A -^ ^^

3. Arbacia e(/g-water.

To obtain the egg-water, the eggs (jf Arbacia were allowed to stand
in little more than their own volume of water for 4£ hours. Given
amounts of Ciona sperm-suspension (see Table XXIII) were added to
(1) 1 cc. plain sea-water, and (2) 1 cc. Arbacia egg-water. The latter
contained the Arbacia eggs, and was not drawn off free of them, as in
previous egg-water experiments. To each of the dishes was then added
10 cc. water, and they were poured on to equal amounts of Ciona eggs.
The experiment was double, being made for two different concentrations
of sperm.

TABLE XXIII. (35.25.4.)

Egg-water

contaiuing

Water Arbacia eggs

_ , ,, \ 3 drops sperm ... 67 0

Oross, A/lj ^ ^Q ^^^p^ ^p^^,^ ^^ ^^^ ^

The complete inhibition of cross-fertilization of Ciona by sperm
treated with Arbacia egg-water, as seen in Table XXIII, was unex-
pected, more especially as A rbacia egg-extract had had the opposite effect.
It was thought that the result might be due to the presence of
numerous Arbacia eggs in the sperm-suspension which was used to
effect the fertilization. The Ciona spermatozoa might conceivably
have become entangled in the jelly surrounding these eggs. A second

284 Studies in tjic Phiisioloyn of Fertilization

possibility was this. The comparatively small \iil\iiiic of water in which
the Arhacia eggs had stooil for a relatively long time may have become
acid enough to inhibit the fertilizing power of the spermatozoa (see
p. 294). The acidity of this egg-water was not tested when it was used
for the experiment, but 1| hours afterwards it gave a pink colour with
a-Naphtholphthalein, whereas normal sea-water gives gi-een.

Owing to these two possibilities of error, the experiment w;is
repeated. After remt)val from the ovary, the Arbacia eggs were washed
by allowing them to settle twice in finger-bowls of water. They were
then placed in 3 — 4 times their volume of water for 55 minutes only.
When this egg-water was di-awn ott' the eggs, it was still green to
a-Naphtholphthalein.

Given amounts of sperm-suspension were added (see Table XXIV)
to (1)3 cc. plain sea- water, and (2) 3 cc. Arbacia egg- water, which was
quite free from Arbacia eggs. 10 cc. of water was added to each, after
which they were poured on to sejjarate equal quantities of eggs. The
experiment was double as last time.

TABLE XXIV. (39.6.5.)

i 3 drops sperm ...
Cross, F/y ^ ,„ , „

(12 drops speiin ...

The Table shows that, under the conditions of this experiment, the
Arbacia egg- water increased the fertilizing power of Ciona sperm in the
same way as the A rbacia egg-extract had don<' in the experiments of
Table XXII.

4. Strong ylocentrotus egg-extract.

The two experiments recorded in this .section were made to test the
action of Strongylocentrotus egg-extract on Ciona sperm, used to cross-
fertilize eggs of the same species. Definite amounts of Ciona sperm -
suspension (.see Table XXV) were added to (1) 10 cc. plain sea-water,

Exp. 1. Bjc

t'ater

Arhacia
ee8-»'ater

10

42

13

77

Exp. 2. (7(/

TABLE XXV. (31.

25

.3.)

Water

Stroiigylo'entrtAu.
egK-extract

, 3 drops sperm ...

■ i-^ ;: :: :.

27

55

02
!)6

81

100

j ■) drops .sperm ...

12 „

•-20 „ „ ...

0
0

83

97

0

100

H. M. FrcHS 285

unci (2) 10 CL-. Strongylocentrotus egg-extract. Each was then poured on
to approximately equal quantities of Ciona eggs. Each experiment was
made triple, three different strengths of sperm being used.

In both experiments, involving six comparisons between the effects
of plain water and of the egg-extract on the fertilizing power of the
Giona sperm, the percentages of eggs fertilized were raised by treatment
with the extract. The results are especially marked in Exp. 2.

5. Strongylocentrotus egg-water.

The first experiment with Strongylocentrotus egg-water was made in
the same way as that with Arhacia egg-water recorded in Table XXIII.
Strongylocentrotus eggs (previously washed twice in finger-bowls of
water) were kept in little more than their own volume of water for "2^
hours. Five drops oi' Ciona sperm-suspension b were added to (1) 1 cc.
normal sea-water, and (2) 1 cc. egg-water, containing Strongylocentrotus
eggs. 10 cc. of water was then added to (1) and to (2), and each was
poured on to separate equal amounts of Ciona eggs A (see Table XXVI).

TABLE XXVI. (.3.5.30.4.)

Egg-water containing
Water Sir'nioytoccnirMus eggs

Cross, A/b ... 27 26

The Table shows that the egg-water had practically no effect on
the fertilization percentages. The conditions of the experiment were the
same as those for the experiment shown in Table XXIII, in which the
Arhacia egg-water inhibited the fertilizing power of the Giona sperm.
That is to say, the egg- water was prepared by keeping the eggs for a
comparatively long time in a relatively small volume of water, and
Strongylocentrotus eggs were present in the sperm-suspension used to
effect the fertilization. At the time of fertilization the egg-water gave
a pink colour with a-Naphtholphthalein, in.stead of the green of normal
sea-water.

For the reasons already given on p. 283 it was thought that
there was in this experiment some other factor influencing the Ciona
sperm than the Strongylocentrotus egg-secretion. A further series of
experiments was therefore made, in which the acidity of egg-water
which has stood for some time on a relatively large volume of eggs was
eliminated, and in which the egg-water used to influence the sperm did
not contain eggs.

Eggs from two females of Strongylocentrotus were washed twice in
finger-bowls of watei-, after which they were placed in tubes with 4 — .5

286 Studies in tli< Physiolofiti of Fertilization

their own volume of watn-. At lengths of time vaiying fnjm 65 — 120
minutes portions of this water were drawn off and used in the experi-
ments. At the time of the last fertilization made, this water was still
green to a-Naphtholphthaleiii. Definite equal amounts of Ciona sperm-
suspension (see Table XXVII) were added to (1) 2 cc. of plain sea-water,
and (2) 2 cc. tif the Strongylocentrotm egg-water, this time not contain-
ing any Strongylocentrotus eggs. 10 cc. water was then added to each',
and they were poured on to separate equal amounts of Gioud eggs. 'I'he
experiments wei'e made double.

TABLE XXVII. (40.16.5.)

troius
Water egg-water

Egg- water 65 minutes on eggs... 3 drops sperm ... 28 77

, 3 drops sperm... 20 73

Exp.

1. Alb.

Exp.

•2. lijc.

Exp.

3. CI<1

Exp.

4. F.jf.

Exp.

r,. F!ci.

Exp.

I!. H/i.

Egg-water 80 minutes on eggs
Egg-water 95 minutes on eggs
Egg-water 120 minutes on eggs
Egg-water 120 minutes on eggs
Egg-water 120 minutes on eggs

) 1 cc. sperm ... 74 9t;

(3 drops sperm... 2fi 65

I 1 CO. sperm . 31 99

( 3 drops sperm ... 60 86

} 1 cc. sperm ... 91 100

\ 3 drops sperm . - 1 73

/ 1 cc. sperm ... 8 79

( 3 drops sperm... 80 lOO

I 1 cc. sperm . 94 100

In all the e.xperiments of Table XXVII the Cioita sperm which had
been treated with Strongylocentrotus egg-water had its fertilizing pow(;r
increased to a marked extent.

As has already been stated, there are two possibilities to account for
the absence of this effect in the experiments of Tables XXIII and
XXVI. It would seem, howevei-, that the possible entanglement of the
spermatozoa in the jelly of the Echinoid eggs present, although it might
conceivably account for the complete inhibition of fertilization in Table
XXIII, could hardly cause no change in the percentages, as seen in
Table XXVI. Considering the .strong inhibitor}' effect of the presence
of small quantities of acid (jn the fertilizing power of spermatozoa, as will
be shown (jn p. 294, it is extremely f)robable that the cause of the
anomalous results obtained in these two experiments was the presence
of acid in the egg- water. It was shown that the egg- water of Table
XXVI gave a pink colour with a-Naphtholphthalein at the moment
when it was used. Thus, jircsuniably owing to the fact that it had

' In Experiments 5 and 6 the sperm was added to 5 ec. (1) water, (2) egg-water, after
which -5 cc. water was added to eacli.

H. M. FiTCHs -287

stood for a considerable time with a large volume of eggs, this egg-
water had an acid reaction, and it is not to be wondered at that this
acidity counteracted the stimulating effect of the egg-water on the
sperm, which latter is clearly shown in the other experiments (Tables
XXIV and XXYII)i.

The end result of the experiments described in the preceding five
sections is therefore that treatment of the spermatozoa of Giona with
egg-extracts of Phallusia, Arbacia or Strong ylocentrotus or with the
egg-waters of the two latter, increases its fertilizing power.

VIII. Further Experiments to test the Specificity of the

Egg-secretions.

The experiments described in the last sections seem to show that the
general specific capability of spermatozoa for fertilizing eggs of the same
species only is not due to their being unstimulated by the egg-secretions
of other species. There remains, however, one other possibility in this
connection. It might be that the specificity of fertilization phenomena
depends on the relatively greater excitation of the spermatozoa by
secretions of the eggs of the same species than by those of others. This
suggestion can be tried by making strict comparisons between the effects
on the fertilizing power of sperm -suspensions produced by extracts from
the eggs of the same and of other species. The hypothesis was
thoroughly tested in the experiments described below.

1. Comparisons of the effects of Strongylocentrotus and of Ciona.
egg-extract on the fertilizing power of Strongylocentrotus sperm.

The chief difficulty encountered in making these experiments is the
practical impossibility of making two different egg-extracts of exactly
equal concentrations. They were made as nearly as possible of the same
strength by taking approximately equal ([uantities of the eggs of the
two species to be experimented upon, crushing them to the same extent,
and then washing off the juices into exactly equal quantities of water.
Diffei-ences of one or two per cent, in the fertilization percentages of
eggs fertilized with sperms treated with two different extracts are
probably to be accounted for by slight differences in the concentrations
of the extracts.

' It should be noted that all other experiments with egg-waters described in this paper
were made under the same conditions as those of Tables XXIV and XXVII, thus eliminating
the acidity factor.

288 Studies in thr Physiohujii of Fertilization

In the first experiment ilu-ce drops of Strongylocentrotus sperm-sus-
pension were added to I'ach of three dishes containing (1) 5 cc. water,
(2) 5 cc. Strongylocentvotus etrg-extract, and (3) 5 cc. Ciona egg-extract.
5 cc. water was tlien addefi to eacli and they were poured on to three
separate equal lots of Strongylocentrotus eggs (see Table XXVIII).

TABLE XXVIII. (4.2.3.6.)

SI rotujt/hvfittrtitus Ciona
Water extract extract

Cross: Strongylocentrotus A/h ... 68 87 88

Both extracts increased the fertilizing power of the sperm In the
same extent.

2. Comparison of the effects of Strongylocentrotus and SphaereclrinKS
egg-extracts on the fertilizing power of Strongylocentrotus sperm.

In each of the two experiments made (Table XXIX), definite equal
amounts of Strongylocentrotus spei-m-suspension were added to (1) 5 cc.
water, (2) .5 cc. Strongylocentrotus egg-exti-act, and (3) 5 cc. Sphaer-
echinus egg-extract. To each was then added 5 cc. water, and they were
poured on to three approximately equal quantities of Strongylocentrotus
eggs. Each experiment was made doid^le. two different strengths of
sperm-suspension being used.

The results of the experiments aix' given in Table XXIX, which

shows that both exti'acts increased the fertilizing pnwer of the sperm

equalh'.

TABLE XXIX (L30.5.)

Exp. 1. SiriiiigijhtreiitiotKK ll/f
Exp. 2. StroiK.niloccMrotus ll/il

3. Comparison of the effects of Strongylocentrotus and Echinus egg-
extracts on the fertilizing power of Strongylocentrotus sperm.

In each experiment definite amounts of Strongylocentrotus sperm
(see Table XXX) were added to (1) 5 cc. water, (2) 5 cc. Strongylocen-
trotus egg-extract, (3) 5 cc. Echinus egg-extract. To each was then
added 5 cc. water, after which they were poured on to three approxi-
mately equal lots of Strongylocentrotus eggs.

The results shown in this Table are not sn unifcirm. In the first
part of Exp. 1 the Echinus egg-extract raised the fertilization percentage

Water

Si,-lHttl]llOVVlU,

extract

mht^

S/)Ii(n'rfchi)\ti:
extract

i •(; ce. spei-m

22

85

S7

( 2 ec. sperm

49

100

100

J -6 cc. sperm

Ul

100

100

/ 2 cc. sperm

88

100

100

H. M. FucHS 289

7 °l^ more than did the Strong ylocenU'otus extract. As the other
three comparisons in the Table show an equal effect of the two extracts
this difference is probably due to an error in the experiment.

TABLE XXX. (2.30..^ and 3.9.6.)

strong
Water e

1 cc. sperm 40 88 95

Strongt/locentrotus Echinus
Water extract extract

■r. , r, ■ . ,, ( 1 CC. sperm

Exp. 1. Stroiuiylocentrotus Ah {

( 3 cc. sperm 62 96 95

■r, , .. , ,,,, ( 3 drops sperm 13 25 26

h,xp. 2. Stroiiqiiloceiitwtus C a ■ ,

^ ^■' ' ( 1 cc. sperm 29 78 77

4. Comparison of the effects of Strongylocentrotus and Echinus egg-
extracts on the fertilizing power of Echinus sperm.

In the following experiment Strongylocentrotus and Echinus extracts
were again used, but in this case a comparison was made between their
effects on the fertilizing power of Echinus sperm. Definite equal
amounts of Echinus sperm (see Table XXXI) were pipetted into
(1) 6 cc. water, (2) 5 cc. Strongylocentrotus egg-extract, and (3) 5 cc.
Echinus egg-extract. To each was then added .5 cc. water, and they
were poured on to separate equal quantities of Echinus eggs. Table
XXXI shows that both extracts had the same effect on the Echinus
sperm.

TABLE XXXI. (-2.31. .5.)

strongylocentrotus Echinus
Water extract extract

Echinus B/c

•6 cc. sperm ... 41 79 78

2 cc. sperm ... 66 84 83

5. Comparison of the effects of Strongylocentrotus and Echinus egg-
extracts on the fertilizing power of Strongylocentrotus sperm, used to
fertilize Echinus eggs.

The experiments detailed in the preceding sections have shown
clearly that the fertilizing power of sperm-suspensions is as much
increased by treatment with egg-extract from another species as it is
by an approximately equal concentration of the egg-extract of the same
form. Evidently then the specificity of fertilization can in no way be
due to any differential effect of egg-secretion from the same and from
other species, and the cause of the general difficulty of effecting inter-
specific hybridizations must be sought for elsewhere.

Now, suppose two species, X and Y, which hybridize with com-
parative ease. Fi'om what has just been said, it would be expected

Journ. of Gen. iv 19

290 Studies in the Physiol (xjn of Fertilization

that tl;e percentage of X eggs fertilized by Y speriii would be increased
to an equal extent by previous treatment of this sperm (1) by X egg-
extract, and (2) by an equal concentration of Y egg-extract. That such
is the case is proved by the following three experiments in which the
cross Echinus microtuherculatus $ x Strong tjlocentrotus lividus </ was
tested, the actions of egg-extracts of these two species being compared.

Although Strong ijlocentrotns sperm of considerably greater concen-
tration is re(iuired to effect hybridization with Echinus eggs, than to
fertilize eggs of the same species, it is by no means necessary to make
use of milky suspensions. Indeed, if such are employed it often happens
that 100"/,^ of the Echinus eggs are fertilized, which is (jf course quite
useless for these comparative experiments.

In each experiment 5 cc. of Strong ijloceiitrdtus sperm-suspension was
pipetted into each of three dishes containing (1) 5 cc. water, (2) 5 cc.
Strongylocentrotus egg-extract, (3) 5 cc. of an approximately equal con-
centration of Echinus egg-extract. Each was then poured on to separate
approximately equal quantities of Echinus eggs. Each experiment was
made double, weaker and stronger sperm-suspensions being used.
Table XXXII shows the results of the experiments.

TABLE XXXII. (3.1.6.)

Strongylo-
ccntrottut Echinus
Water extract extract

„ , _ ,. , ,„ , ( Weaker sperm ... '2-2 32 31

^ ' ■'■' \ Stronger sperm... .58 71 70

^ , „ ,. , ( Weaker sperm ... 12 40 39

txp. 2. Echttius A/StronqiiloceiUrottis n < „

^ ' ••' ( Stronger sperm... 0.5 08 70

_,„_,. , ,„ , ( Weaker sperm ... 40 70 70

Exp. 3. Echinus A/Strunriiilocentrotus p <„

' ■• ' I Stronger sperm... 72 71 73

In all the experiments except the second part of Exp. 3, both
extracts raised the percentages of eggs hybridized by an equal amount,
allowing for 2°/^ experimental error. That there was no raising of the
percentages in the second part of Exp. 3 is explained by the fact that a
sample of the Echinus eggs A, fertilized with Echinus sperm in plain
water, showed only 74 7o of segmenting eggs. Evidently there was a
considerable number of unfertilizable eggs present, and the maximum
possible percentage had been touched on in the fertilization by untreated
sperm in the second part of Exp. 3.

Although the experiments described in the last five sections have
shown that the hypothesis which they were made to test is unten-
able, the results given in Table XXXII indicate a valuable method of

H. M. FocHs 291

increasing the number of eggs fertilized in inter-specific hybridization,
namely, the preliminary treatment of the sperm with egg-extracts.

As has already been mentioned, the sperm-suspension used in the
hybridization experiments of Table XXXII were not very concentrated.
Nevertheless, they were strong enough to show under the microscope
hales of spermatozoa collected round the eggs in the second parts of the
last two experiments. A comparison of the size of these halos is of
interest. In Exp. 2, halos were not visible round the eggs fertilized by
the water-suspension of sperm. They were present, however, both
round the eggs inseminated by the Stroiif/i/locetitrotus extract suspension
and by the Echinus extract suspension, and they were of equal size in
each. In Exp. 3 there was a slight zone of spermatozoa visible round
the eggs fertilized with the water-suspension, and strong, equally large
ones surrounding the eggs in the sperm-suspensions made up in the
two extracts.

6. Comparison of the effects of Strongylocentrotus and Asterias egg-
extracts on the fertilizing power of Strongylocentrotus sperm.

Von Dungem (1, p. -36) found that a substance can be obtained
from the eggs oi Asterias by breaking them up, which in small doses is
poisonous to the spermatozoa of Echinoids, but which does not affect
the sperm of the same species. The Echinoid spermatozoa could be
seen under the microscope to be killed by this substance. The latter is
given off by the eggs into the sea-water, and owing to its presence the
spermatozoa of Echinoids cannot enter the eggs of Asterias.

This discovery appeared to me to be the more surprising as my
previous experiments had shown no such phenomena for the forms
experimented with. It was desirable, therefore, to repeat Von Dungern's
experiments with the Asteroid extract. This was earned out, using the
same strictly comparative method employed in the other experiments
described in this paper.

The first trials were made with egg-extracts obtained in the usual
way. Definite equal amounts of Strongylocentrotus sperm-suspension
(the quantities are given in Table XXXIII) were added separately to

TABLE XXXIII. (3.20.6.)

StrongylocentTotus A stcrias
Water extract extract

„ , „ , , , ( 3 drops sperm 8 68 0

Exp. 1. Stronnylocentrotus dih { ^ „, ,„„ „

^ •"' ( 1 ec. sperm ... 62 100 0

„ „ „ , , , \ 3 drops sperm 28 89 0

txp. 2. btrongmocentrolus Ac { ^ ., , ,„, „

' ( 1 CO. sperm ... 84 100 0

19-2

292 Studies in the Plijisiolofin of Fertilization

(1) 5 cc. water, (2) 5 cc. Stroiiffi/lnceiih'utiis egg-extract, (3) 5 cc. Asterias
glacialis egg-extract. 5 cc. water was then added to each, and they
were added to separate ei|ual ainmints of Stronf/i/locentrutus eggti. Each
of the two experiments was dinible, two strengths of sperm being used.

The results given in the Table show that the Asterias glacialis egg-
extract had the effect described by Von Dungern. Whereas the Stronyy-
locentrutus extract increased the fertilizing power of the sperm of that
form (as was previously shown in Table XII), the Asterias extract com-
pletely paralyzed the Strong ylocentivtus sperm, so that it was incapable
of fertilizing a single egg.

7. Comparison of the effects of Strongylocentrotus and Asterias egg-
waters on the fertilizing power of Strongylocentrotas sperm.

It remained, finally, to see whether this poison for Strong yloantrutus
sperm is given off by the eggs into the water or not. Egg-waters of
Strongylocentrotus and Asterias were made up of approximately equal
concentrations as follows. The eggs of the two forms were washed
thoroughly by allowing them to settle four times in finger-bowls full of
water. They were then placed in tubes in seven times their volumes
of water. After li hours the waters were drawn off the eggs and used
in the following experiment.

Definite equal amounts oi Strongylocentrotus spcrni-su-spension were
added to (1) 2cc. water, (2) 2 cc. Strongylocentrotus egg- water, and
(3) 2 cc. Asterias egg- water. 10 cc. of water was then added to each
and they were poured separately on to approximately equal lots of
Strongylocentrotus eggs. The experiment, the result of which is detailed
in Table XXXIV, was made with two different amounts of sperm.

TABLE XXXIV. (4.2.3.G.)

^ , , , ( 3 drops sperm ..

Cross: Stronqiilocentrotus Ac

( 1 cc. sperm

The result shows clearly that the poison is not given off by the
Asterias eggs into the water. On the contrary, the Asterias egg-
secretion stimulates the Strongylocentrotus spermatozoa to just the
same extent as does the Strongylocentrotus egg-secretion itself. I can
only explain Von Dungern's statement to the contrary on the sup-
position either that broken eggs were present among those from which
he obtained the egg-watei', or that some of the mucus from the outside
of the body of the Asteroid was present with the eggs. Von Dungern

Slronifytoc^ntrotits
i'ater egg-water

Asterias
eggwater

4 18

17

30 70

71

H. M. FucHS 293

himself has shown that the latter also contams a poison for Echinoid
spermatozoa.

The result of the experiment described last shows that the effect of
Asterias egg-secretions on the sperm of other species is no different
from that of the other forms experimented with. The presence of a
substance in the Asteroid eggs which is poisonous for Echinoid sperma-
tozoa is interesting in itself but it cannot be an example of the general
way in which eggs protect themselves against fertilization by sperm of
other species. For if this were so, the extract of Giona eggs would not
have an equally stimulating effect on the spermatozoa of such a widely
different form as Strong ijlocentrotus as has the egg-extract of the latter
species itself

IX. Effects of the H-ion Concentration of the Water on
THE Fertilizing Power of Sperm.

In the experiments to be described in the two following sections
comparisons were made between the fertilizing powers of sperm-sus-
pensions of Cionu of equal concentrations made up (1) in normal sea-
water, and (2) in sea-water of altered H-ion concentration. The
experiments were carried out on exactly the same lines as those already
described on the effects of egg-secretions on the sperm. It may be
stated at once that the results showed an increase in the fertilizing
power in the presence of a slight decrease in the H-ion concentration,
and a very marked inhibition for a small increase in the latter.

1. Effect of a decrease in the H-ion concentration.

In each of the two experiments, the results of which are shown in
Table XXXV, two drops of a sperm-suspension of Giona were added to
each of the two dishes containing (1) 100 cc. normal sea- water, (2) 100 cc.
alkaline sea-water. The latter was made up by the addition of 1 cc.
N/10 NaOH to 200 cc. sea- water. 10 cc. of each of the suspensions
(1) and (2) were then poured on to separate approximately equal
quantities of eggs ft'om another individual of Giona.

TABLE XXXV. (30.1.3.)

Normal water Alkaline water

Exp. 1. A/b 0 9

Exp. 2. C/d 0 24

Thus in both cases the effect of the alkaline water was to increase
the fertilizing power of the sperm-suspension.

294 Studies in the F/ii/.siolo(ji/ of Fertilization

2. Efied of an increase in the H-ion concentration.

In the first series of experiments equal quantities (given in
Table XXXVI) of a Ciona sperm-suspension were pipetted into
(1) 100 CO. normal sea-water, (2) 100 cc. acid sea-water, the constitution
of which is given below. 10 cc. of each of the liquids (1) and (2) were
then poured on to separate approximately (M|ual amounts of eggs of
another individual Ciona. Exj). 1 was made for two, and Exp. 3 for
three different strengths of sperm.

The acid sea-water.s (2) were made up as follows:

In Exp. 1, •2cc. N/10 HCl added to 100 cc. .sea- water.
In Exp. 2, ■:]
In Exj]. 3, 1

Each of these solutions was tested previously, using the colorimetric
method elaborated by Sorensen (4). The addition of eight drops of
a-Naphtholphthalein to test-tubes containing 10 cc. of the liquid gave
the following colours:

Normal sea-water Green.

Acid water of Exp. 1 V. light gi-een.

Ditto of Exp. 2 Light greenish briiwn.

Ditto of Exp. 3 Pink.

The results of these experiments are given in 'I'able XXXVI.
TABLE XXXVL (.10.14.3.)

Normal water Acid water

Exp.i. K/r \ f- "''™ ■ ° "^

'• ( 20 cc. sperm ... 100 8

Exp. 2. C/d 5 cc. sperm ... 3 0

f -Ct cc. sperm ... 45 0

Exp 3. A/b I 2 cc. sperm ... 97 0

' 5 ce. sperm ... 100 0

In the first part of Exp. 1 too little sperm was added to give a com-
parison. In the second part of the same experiment the effect of the
acid water on the sperm was to cause a great fall in the fertilization
percentage. In Exps. 2 and 3, of which Exp. 2 was made with a slightly
greater and Exp. 3 with a con.siderably greater concentration of H-ions
than Exp. 1, the effect of the acid waters on the sjxn-matozoa was to
inhibit fertilization completely.

H. M. FucHS 295

One further experiment was carried out, in which a comparison was
made between the effects of normal water and t)f three increasing con-
centrations of acid water on the fertilizing power of a sperm-suspension
of Giotm., used to effect a particular cross. 1 cc. of sperm-suspension h
was pipetted into each of four dishes containing (1) 100 cc. normal sea-
water, (2) 100 cc. acid water A, (3) 100 cc. acid water B, (4) 100 cc. acid
water C. 10 cc. of each was then poui'ed on to separate approximately
equal quantities of Ciona eggs G.

The acid waters were made up as follows :

A. -2 cc. N/10 HCl in 100 cc. sea-water (V. light green

with ct-Naphtholphthalein).

B. •4cc. N/10 HCl in 100 cc. sea-water (Light brown

with a-Naphtholphthalein).

C. -6 cc. N/10 HCl in 100 cc. sea-water (Light pinkish

brown with «-Naphtholphthalein).

The result of the experiment is given in Table XXXVII, which
shows that in this case even the least increase in the H-ion concen-
tration of the water completely inhibited the fertilizing power of the
sperm-suspension.

TABLE XXXVIL (31.1.5.3.)

Normal water Acid water A Acid water B Acid water C
Cross : Glh 89 0 0 0

The first experiment on the effect of Arhiicia egg- water on Giona
sperm (Table XXIII) showed inhibition of the fertilizing power of the
latter, and the first experiment on the influence of Strongylocentrotus
egg- water on Ciona sperm (Table XXVI) showed no change in the fer-
tilizing power. It was seen that on the repetition of these experiments
with egg-waters whicli had stood for a shorter time with a relatively
smaller quantity of eggs, the fertilizing power of the sperm was
increased in the same way as it was by treatment with the egg-extracts.
It was suggested that the results obtained in the experiments' of Tables
XXIII and XXVI were due to the presence of acid in the egg- waters.
The egg-water used in Table XXVI gave, at the time fertilization was
made, a pink colour with a-Naphtholphthalein (p. 2S5), whereas the
normal sea-water gave green. Tables XXXVI and XXXVII show that
a lesser increase in the concentrations of H-ions in the water than is
enough to give a pink colour with the indicator will quite inhibit the
fertilizing power of sperm-suspension of Ciona, — at any rate of suspen-
sions of about the strength of those used in the experiments described

296 Studies in the Physiology of Fertilization

in this paper. Thus it is extrcnit'ly probable that tlie results of Exps.
XXIII and XXVI arc to be put down to this cause.

3. The reactions of Ciona and Asterias egg-extracts and of Ciona
blood.

Since spermatozoa show themselves to be so very sensitive to small
alterations in the H-ion concentration of the water, it is necessary to
test the reactions of the egg-extracts and egg- waters. For if the latter
showed a lesser H-ioii concentration than that of normal .sea- water, their
stimulating effects on spermatozoa might be due to this cause.

With a view to settling this, comparative colour tests were carried
out with normal sea-water and Ciomi egg-extract, the latter of about
the same concentration as that used in the experiments. Three
indicators were used\ as is shown in Table XXXVIII.

TABLE XXXVIII. (2.11.6.)

Normal water Ciona extract

a-Naphthol|ibthaleiu, 8 drops in 10 cc. Green Green

Neutral Red, 2 drops in 5 cc. ... Yellow Yellow

Phenolphtlmlein, 12 drops in 5 cc. ... Pink V. faint pink

The first two indicators showed no difference between the reactions
of the normal water and of the water containing egg-extract. The
phenolphthalein, however, indicated that the extract had a slightly
greater concentration of H-ions than the water. If this difference had
been in the other direction, it might have explained the effect of the
extract on the fertilizing power of sperm. As it is, however, the cause
of this must be sought in some other factor, which remains at present
undetermined.

As the extract of the eggs of A.iterius had a reverse effect on sperm-
suspensions from the other extracts examined, its reaction was specially
investigated with the colour indicators. The results of the tests are
given in Table XXXIX.

TABLE XXXIX.

Normal water

AskriaA extract

a-Naphtholphthalein ...
Neutral Red

Green
Y'ellow

Faint green
Piuk

' Too much reliance cannot be put on the colour reactions given in this section, since
Soreusen, Ergehnisse der Phi/siohgit'. states that the indicators used, namely Neutral
Red, a-Naphtholphthaleiu and Phenolphthalein, are not always trustworthy in the
presence of proteins. Neutral Red is said to be "kaum brauchbar," and the other two
"nur selten."

H. M. FucHS 297

Both indicators show that the water containing the Asterias extract
has a considerably greater concentration of H-ions than normal sea-
water. The colour given by the Asterias extract with a-Naphthol-
phthalein approximates to that given by the "acid water" of Exp. 1,
Table XXXVI, and by "acid water A" of Table XXXVII, both of
which inhibited the fertilizing power of Giona sperm-suspensions. If
one assumes a like action on Strung tjlocentrotus sperm, the " poison " for
the latter which is contained in A sterias eggs may be the presence of
an acid. This is, however, only a suggestion which remains quit(>
unproved.

Finally, colour tests were made with the blood of Ciona to see
whether the change in its effect on the fertilizing power of Ciona sperm,
according to the time the animals from which it is taken have been in
the Aquarium, is correlated with any change of reaction. It will be
remembered (see p. 281 seq.) that the blood of animals taken fresh
from the sea increased the fertilizing power of sperm-suspensions, while
a sojourn of the animals for even one night in the Aquarium water
reversed the etfect of the blood on the sj^erm.

For the colour tests the blood was first centrifuged in order to obtain
the liquid fairly clear and colourless. The plasma showed a red colour
with Neutral Red, yellow with Methyl Orange and pink with a-Naph-
tholphthalein. There was no alteration in the reaction when the
animals had been kept in the Aquarium, the latter experiments being
made with animals from the lot used in Series D and E, Table XIX.

The fact that the blood gave a pink colour with a-Naphtholphthalein
shows that it has a concentration of H-ions considerably greater than
that of normal sea-water. Tables XXXVI and XXXVII showed that if
acid is added to the latter until it gives a pink with this indicator, the
fertilizing power of sperm-suspensions made up in it is completely
inhibited. From this it follows that there must be something in the
blood of fresh Ciona which ct)unteracts the effect of this acidity as well
as stimulates the spermatozoa. It might be added as a suggestion that
if this stimulating substance disappeared from the blood after the
animal had been kept in the Aquarium water, the acidity of the blood
would have the effect of paralyzing the spermatozoa, which would be a
possible explanation of the observed phenomena.

298 Studies In the Phj/Kiolugn of Fertilization

X. Conclusion.

Soon Jifter the prefsent work was commenced a pioliminary .•tccount
of an investigation by F. R. Lillie published some months previously (2)
came to my notice. Before my experiments were completed the full
account of this very important work was published (3). It consisted of
a study of the reactions of spermatozoa to substances secreted by the
eggs, and was thus concerned with the same general problem as the one
I was attacking. Lillie's methods, were, however, quite different from
mine. The main results were based on observations of the activity of
the spermatozoa made both with the naked eye and more especially
under the microscope, when the spermatozoa were treated with egg-
extracts, substances secreted by the eggs, and chemical agents. Besides
this, the effects of the treatments on the subsequent capability of the
sperm for fertilizing eggs were tried. Thus, whereas Lillie's results
depended mainly on direct observation, mine were based on indirect
methods. The effects of the different factors on the spermatozoa were
in my case investigated by comparing the subsequent fertilizing powers
of the suspensions of sperm experimented upon. The main result of
Lillie's work was to show that substances are secreted by the eggs of
Arbacia and of Nereis which cause intense activity of the spermatozoa,
agglutinate them in masses and to which the spermatozoa are positively
chemotactic.

Now Lillie found evidence for a specificity in the egg-secretion.s.
The Nereis "agglutinin" did not agglutinate Arbacia sperm, but the
Arbacia substance was agglutinative for Nereis sperm. The Arbacia
extract, however, probably contained two agglutinins, one specific for
the Arbacia sperm and the other not specific ; for if the substances from
Arbacia eggs were kept for some days, Nereis sperm was no longer
agglutinated, while Arbacia sperm was. Moreover, the coelomic fiuid
of Arbacia contained an agglutinin for Nereis sperm, which did not
affect Arbacia sperm.

It will be remembered that I could find no evidence for specificity
in the effects of egg-secretions on the fertilizing power of the sperm.
On the contrary, in all the forms tried, the fertilizing power of a sperm-
suspension was increased to an exactly equal extent by equal concentra-
tions of the egg-secretions of the same and of another form, the two

H. M. FucHS 299

forms being sometimes as widely different as Ascidians and Echinoderms.
The factor in the egg-secretions, then, which increases the fertilizing
power of the sperm, has no connection with the specificity of the fertili-
zation process. It may well be that this specificity is due to the " iso-
agglutinins" of Lillie — that for a spermatozoon to fertilize an egg, there
must be a reaction between it and the iso-agglutinin of the egg, the
visible effect of which is agglutination.

In the one point in which Lillie's experiments and my own coincide
there is complete disagreement. All my experiments have shown that
treatment of a sperm-suspension with egg-secretions, or with substances
artificially extracted from the eggs, increases the fertilizing power of the
suspension — that after this treatment more spermatozoa can fertilize
eggs than before the treatment. Lillie, however, states that there is a
loss or diminution of the fertilizing power of the sperm as an effect of
the egg-extract. Special experiments were made on the exact lines as
those of Lillie (see p. 275), but they too gave the invariable result, — the
fertilizing power was increased. I am therefore at a loss to explain this
after-effect on the sperm described by Lillie for his experiments.

The end result of my own investigations is that eggs of Ascidians and
of Echinoids secrete substances into the sea-water in which they lie.
These substances increase the fertilizing power of sperm-suspensions.
If a sperm-suspension of a certain concentration be taken and divided
into two equal portions, to one of which is added some plain sea-water,
and to the other an equal amount of sea-water containing egg-secretions,
the latter portion can fertilize more eggs than can the former. Further,
the egg-secretions of a foreign species increase the fertilizing power to
an exactly equal extent as those of the same species, provided they are
equally concentrated. This increased fertilizing power of sj)erm-suspen-
sions was shown for a number of ordinary intra-specific cross-fertilizations
and for one case of hybridization.

In conclusion, it should be pointed out that the conditions in nature
must be somewhat different from those in the laboratory. For as the
substances in question are secreted by the eggs lying in water, the
secretions must be continually washed away by currents and wave
action. It follows that at a small distance from the surface of the egg
the secretions will be present in the water only in minimal concentra-
tions. The action on the spermatozoa must therefore take place on, or
at a very small distance from, the surface of the egg.

300 Studies in (lie Phyxiolvf/n of Fertilization

XL Summary of Experimental Results.

1. It" a certain number of eggs of an individual A nf Cioun, in
a given aniount of plain sea-water, are fertilized l)y the addition of
a certain quantity of a sperm-suspension h of another individual, fewer
eggs segment than when approximately the same number oi A eggs in
the same amount of sea-water, but this time containing egg-extract, are
fertilized by an equal amount of sperm-suspension h.

2. The rate of segmentation of the eggs is the same in jilain water
and in egg-extract.

3. The extract has an immediate effect in raising the fertilization
percentage.

4. The phenomenon described in (I) is the same in the case of
Ascidia.

5. Ovary-extract of Ciona has the same effect as egg-extract.

6. In Ciona the fertilization percentage is raised by exactly the
same amount by equal concentrations of extract derived from the same
animal as the eggs used in the cross, from the same animal as the sperm,
and from a third animal.

7. The extract acts as a aperiir " stimulant " in Ciona and does not
affect the eggs. Its presence in a sperm-suspension of a certain concen-
tration causes more spermatozoa to fertilize eggs than is otherwise the
case, i.e., it increases the " fertilizing power " of the sperm.

8. The sperm of Strong ijlocentrotus is affected by the egg-extract of
the same .species in exactly the same way as is that of Ciona.

9. In Stvongylocentrotus the stimulating action of the extract con-
tinues as long as the sperm-suspension is capable of effecting fertili-
zation.

10. The eggs (if Ciomt, Arbacia. and Strdiigi/locentrotus secrete a
substance or substances into the water which increase the fertilizing
power of the sperm of the same species, in the same way that the egg-
extracts have been shown to do.

11. The effect of the blood of Ciona on the fertilizing power of the
sperm depends on the time that the animals from which the blood
is taken have been in the Aquarium. The blood of freshly caught
animals increases the fertilizing power of the sperm, while that of

H. M. FucHS 301

animals which have been in the Aquarium fur one night or longer
decreases it.

12. Egg-extracts of Phallusia, Arbacia and Strongylocentrotus and
egg-secretions of the two latter increase the fertilizing power of sperm-
suspensions of Giona.

13. The fertilizing power of a Strongylocentrotus sperm-suspension
used to fertilize eggs of the same species, is increased to the same extent
by egg-e.xtracts of Strongt/locentrutus, Sphaerechinus, Echinus and Ciena.
The fertilizing power of an Echinus sperm-suspension used to fertilize
the eggs of that form is increased to an equal extent by the extracts of
both these forms.

14. Asterias egg-extract completely inhibits the fertilizing power
of a Strongylocentrotus sperm-suspension. The egg-secretion of Asterias,
however, increases the fertilizing power of the latter to the same extent
as does Strongylocentrotus egg-secretion itself.

15. A small rise in the H-ion concentration of the water gives an
increase in the fertilizing power of a sperm-suspension of Giona; while
a small fall causes a very marked decrease in the latter.

16. Water containing Giona, egg-extract has a very slightly greater
concentration of H-ions than normal watei'. Asterias extract, however,
has a considerably greater concentration. The blood of Giona shows no
change in reaction con-elated with its changed effect on the fertilizing
power of sperm-suspensions.

REFERENCES.

1. Vox DuNGERN, E. "Neue Versuche zur PhysiologiederBefruohtung." Zeitschr.

f. ally. Physiol. Bd. i. 1902, S. .34.

2. LiLLiE, Frank R. "The production of sperm i.so-agglutinins by ova." Science,

Vol. xxxvi. 1912, p. 527.
3. "Studies of Fertilization." V. /oura. E/;;^ .^oo^. Vol. xiv. 191.3, p. 515.

4. SoRENSEN, 0. R. C. R. Lab. Carlsbery, Tom. viil. 1909, p. 1. Also Biochem.
Zeitschr. Bd. xxiv. 1910.

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This series recently established by the Trustees of the University owes its orif;in to a
feeling that there should be a medium of publication occupying a position between the
technical journals, with their short articles, aud the elaborate treatises which attempt to
cover several or all aspects of a wide field. The volumes of the series will differ from the
discussions generally appearing in technical journals in that they will present the complete
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in scattered articles, if published at all. On the other hand they will differ from detailed
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the subject in as summary a manner and with as little technical detail as is consistent
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THE FIRST PUBLISHED VOLUME OF THE SERIES

The Evolution of Sex in Plants. By John Mehle Coulter, Head of
the Department of Botany in the University of Chicago, viii + 140
pages, small 12mo, cloth; 4s. net.

In this first volume of the new "University of Chicago Science Series" Professor
Coulter, the editor of the Botanical Gazette and the author of numerous volumes on
botanical science, has given a presentation of the results of research showing that all
reproduction is the same in its essential features and all methods of reproduction are
natural responses to the varying conditions encountered by plants in their life histories.
Sex reproduction, the author says, is simply one kind of response, the sex feature not
being essential to reproduction, but securing something in connection with the process.
Various phases of the subject discussed include the evolution of sex organs, the alternation
of generations, the differentiation of sexual individuals, and parthenogenesis. The last
chapter, which offers a theory of sex, reviews the more prominent facts set forth in
preceding parts of the volume, and serves both as a summary and a working hypothesis.

OTHER VOLUMES IN PREPARATION FOR EARLY PUBLICATION

The Origin of the Earth. By Thomas C. Chamberlin, Head of the
Department of Geology in the University of Chicago.

The Isolation and Measurement of the Electron. By Robert
Andrews Millikan, Profes.sor of Physics in the University of Chicago.

Finite Collineation Groups. By Hans F. Blichfeldt, Professor of
Mathematics in Leland Stanford Junior University.

OTHER VOLUMES PLANNED AND IN PREPARATION
The Evolution of Reptiles. By Samuel Wendell Williston.
Food Poisoning. By Edwin Oakes Jordan.
The Problem of Individuality in Organisms. By Charles Manning

Child.
The Development of a New System of Organic Chemistry,

Based on Dissociation Concepts. By John Ulric Nef, with

the co-operation of J. W. E. Glattfeld.
The Living Cycads. By Charles Joseph Chamberlain.
Mechanics of Delayed G-ermination in Seeds. By William Crocker.
The Rigidity of the Earth and of Materials. By Albert A.

Michelson.
The Problem of Fertilization. By Frank R. Lillie.
Linear Integral Equations in General Analysis. By Eliakim

Hastings Moore.

The University of Chicago Press

The Cambridge University Press

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London, Fetter Lane

Volume IV APRIL, 1915 No. 4

NOTE ON THE INHERITANCE OF HETEROSTYLISM
IN PRIMULA ACAULIS J ACQ.

By R. p. GREGORY, M.A.

Fellow of St John's College, Gamhridge; University Lecturer in .^

Botany. ,jj

The experiments with the wild Priimose, which it is the purpose of aAKUK"-^*
this note to record, were begun by Mr Bateson and the writer con-
currently with our exj)eriments on Primula sinensis^. The results
obtained in the two species are exactly similar ; the inheritance of the
characters of short and long style is of a simple Mendelian type, the
short style being dominant, the long style recessive.

The two forms of the wild Primrose are about equally numerous in
nature-. Among the wild plants used for experiment were nine short-
styled plants, all of which proved to be heterozygous. Darwin' found
the " illegitimate " mating short-style x short-style to be relatively even
less fertile in the Primrose than it is in P. sinensis; my experiments
have given a similar result, and from numerous matings of this kind
only five families have been obtained ■*. Two of these families were the
offspring of wild plants ; they consisted of 13 short-styled, 4 long-
styled plants. Of these 13 short-styled plants, only two produced any
offspring ; one of them shewed itself to be heterozygous, giving 1 1 short-

' Bateson and Gregory, Roy. Soc. Proc, B. Vol. Lxxvi. p. 581, 1905 ; Gregory, Journal
of Genetics, Vol. i. p. 73, 1911.

2 Darwin, Forms of Flowers, p. 34. I have counted the two forms in several localities
where the plant grows wild, without finding any significant departure from equality of
numbers.

» L.C., p. 37.

* The plants used for experiment were grown out-of-doors, in pots covered with muslin
bags, ao that flowers which had been operated upon were exposed to the weather at a time
of year when frosts are common. As a consequence a great number of the experiments
were unsuccessful, and the whole of the crosses made in 1905, and again those made in
1907, were lost.

Journ. of Gen. iv 20

304 Heterostylisni in Primula

style and 4 long-style when self-fertilized, and 9 short-style, 1 1 long-
style, when crossed by the recessive. The other short-styled plant,
when crossed by long-style, gave 4 short-style, 0 long-style ; other
experiments with this plant failed, so that '\i remains doubtful whether
it was pure or heterozygous. It was the only short-styled plant, from
which seeds were obtained, which was not definitely shewn to be
heterozygous. Altogether, the heterozygous short-styled plants, self-
fertilized, gave 39 short-st3'le, 13 long-style. The crosses of hetero-
zygous short-style % x long-style (^ gave 1 1 9 short, 138 long ; the
reciprocal crosses gave 110 short, 96 long; or a total, for the matings
in both forms, of 229 short, 234 long. The results of my experiments
are shewn in tabular form below.

Form of Mating

dumber of
Families

Short-style

Long-style

Expectation

Long X Long

21

0

199

All Long

Short X Short (heterozygous) ...

5

39

13

3D : IR

Heterozygous Short 9 x Long i

17

ll!t

138

W -.IR

Long ? X Heterozygous Short <J

15

110

oe

ID : IR

ON VARIEGATION IN PRIMULA SINENSIS.

By R. p. GREGORY, M.A.

Fellow of St John's College, Cambridge; University Lecturer in

Botany.

CONTENTS.

PAGE

Introduction . . 305

The plastids of the green aucl of the yellow (yellowish-yreen) cells . . 307

The variegated and yellow-leaved plants ....... 308

Breeding Experiments 311

Discussion of an hypothesis ......... 313

Description of Plates 320

Introduction.

The purpuse of this paper is to describe some observations upon
a race of Primula sinensis, in which it has been found that the alter-
native characters of normal green, variegated or pale yellowish-green
leaves (and other organs containing chloroplasts) are transmitted from
parent to offspring through the egg-cells only, the male gamete playing
no part in determining the nature of the zygote in respect of these
characters.

The experimental results which have been obtained are similar to
those which have already been described by Correns' in Mirabilis
Jalapa albomaculata, and by Baur- in Antirrhinum majus alhomacu-
latum, the principal diflerence being that, whereas in Mirabilis and

' " Vererbungsversuohe mit blass (gelb) griineu und buntblattrigen Sippen bei Mirabilis
Jalapa, Urtica pilulifera und Lunaria annua," Zeitschr. /. Induktive Abstammungs- u.
Vererbungslehre, i. p. 291, 1909 ; " Zur Kenntnis der RoUe von Kern und Plasma bei der
Vererbung," Ibid. n. p. 331, 1909.

- "Untersuchungen iiber die Vererbung von Chromatophorenmerkmalen bei Melan-
clrium. Antirrhinum und Aquilegia," Zeitschr./. Induktive Abstammungs- u. Vererbungs-
lehre, IV. p. 81, 1910. In this paper (p. 99) Baur records the fact that he has observed
the maternal inheritance of variegation in Primula sinensis, without, however, studying
it fully.

20—3 >M

306 Varicf/atioii in Primula sinensis

Antirrhinum the yellowish-white plants, which are entirely devoid of
normal chloroplasts, do not survive beyond the seedling stage, in
Primula it has been found possible to raise a few examples of the
corresponding ])ale-coloured typo to maturity and to use them both as
the female and as the male parents in crosses.

The pale yellow or yellowish-green plants oi Primula differ from the
normal green type in having smaller chloroplasts, which are of a pale
yellowish-gi'een colour, instead of being bright green. There is some
variation among the yellow-leaved plants as regards the degree of
chlorosis'; some are very pale, others are a greenish -yellow, the
differences in appearance being due to difference in the size and pig-
mentation of the chloroplasts. The variegated plants consist of a
patchwork of cells of two kinds, containing respectively bright green
and pale-c(jlour(xl chloroplasts. In the mature organs of the variegated
plants, the individual cells, at any rate in the great majority of cases,
contain chloroplasts of one kind only, but evidence has been obtained
that in the very young leaves two kinds of chloroplast may be present
together in the same cell. The bearing of this observation on the
genetic problem of the maternal transmission of variegation is dis-
cussed on pp. 314-317.

As in Mirabilis and Antirrhinuni, the \ariegated plants oi Primula,
when self-fertilized, give three kinds of offspring, namely, self-coloured
green, variegated and self-coloured yellow, in irregular proportions.
The green offspring of a variegated plant give only green progeny in
succeeding generations, the variegated continue to give all three types.
Most of the yellow-leaved plants die at an early stage, but a few indi-
vidtials have been brought to flower; I have not succeeded in obtaining
seeds from the self-fertilization of these plants, but they have been
crossed with normal green plants. The result of these experiments is
to shew that, apart from any question of variegation, the character of
the chloroplast is transmitted through the egg-cell only ; when the
yellow plant is used as the female parent all the offspring are yellow ;
when it is used as the male parent, in a cross with a green plant, both
the Fi and the succeeding generations consist entirely of self-coloured
green plants, the yellow character not having been transmitted to the

' As the term "chlorosis " in some sense connotes disease, it should be said that the
term is used in this paper in a purely descriptive sense, applied to a condition of the
plants. The chlorosis in Primula is inherent in the plant and is not due to methods
of culture. Nor has it anything to do with the "infectious chlorosis" of some plants (see
Baur, loc. rit.).

R. P. Gregory 307

offspring through the pollen of the male parent. This result con'e-
sponds with the similar results obtained by Correns and Baur when
crosses were made with flowers taken from the j)ure white (yellowish-
white) shoots sometimes bonie by the variegated plants of Mirahilis
and Antirrhinum.

It is to be noticed that crosses between green and yellow plants do
not give variegated heterozygotes ; all the oft'spfing are pure for the
character borne by the female parent used in the cross. The original
variegated plant, from which my variegated race has been bred, appeared
in the F„ of a cross between two normal green races, Ivy-leaf and Snow-
drift" ; with this exception, variegated plants have invariably been the
I >ffspring of a variegated mother.

As might be expected, the character of the chloroplast is found not
to be affected by the presence or absence of anthoeyanic pigment in the
cell-sap. The original variegated race was without sap-colour; it has,
however, been brought in through matings with other races, and the
various combinations of green and of yellow plastids with sap-colour
have been obtained in the progeny.

The variegated character of the stems and leaves, again, is quite
independent of the flaked or striped flowers, which result from the
development of sap-colour in some cells and its absence from others.
The two characters, in fact, stand in contrast to one another, for the
flaked character of the flowers is inherited through the male, as well as
through the female parent. Here again my results agree with those
described by Con-ens in Mirahilis-.

The plastids of the green and of the pale yelloiu or yellowish-green cells.

In the fully grown organs of the plant, the plastids contained in any
individual cell are, in general, of one kind only; those of the chlorotic
cells are smaller than those of the green cells and are of a pale
yellowish-green colour, instead of being bright green. The difference
between the normal and chlorotic plastids is very strikingly shewn if a
variegated plant be examined after exposure to bright sun-light ; the
plastids of the normal cells are then packed with rounded or oval
starch-grains, while those of the neighbouring chlorotic cells contain
only a few small granules of starch (PI. X, iig. 7). The differences in the

' A description of these races is given in Journ. of Genetics, Vol. i. p. 102, 1911.
- Zeitschr.f. Induktive Abst.- u. Vererbungslehre, i. p. 322, 1909.

308 Varief/ation in Primula sinensis

size of the plastids persist after tiie starch has been removed by keeping
the plants dark for some days (PI. X, iigs. 3 — 6).

In the young leaf of the variegated or yi^llow plants the ])lastids of
the chlorotic cells are very small and almost colourless (?1. X, figs. !), 13) ;
in the older leaves they have increased in size (PI. X, fig. 2) and their
pigment is obvious, though they always remain smaller and paler than
the typical green plastid. Cells have been found containing plastids
which exliibit intermediate degrees of chlorosis (PI. X, figs. 8,10); plastids
of this kind are rather larger than in the extreme case and they form
lai'ger quantities of starch, though not so nnu-h as do the normal green
plastids.

The variegated and yellow-leaved plants.

The variegated plants oi Pnviida sinensis shew \ery much the same
series of forms as those described by Correns in Mirahilin Jaliipa albo-
macidata. The green and yellow cells are mingled in a mosaic, which
may be finely divided, small groups of cells of one kind forming tiny
flecks scattered among cells of the other kind; or may be coarse, cells
of one kind forming patches of considerable size, or even whole leaves
or sectors of the plant. When the mosaic is coarse, the component parts
may be quite irregularly arranged, but generally there is a tendency
towards the formation of a pattern, which, however, is usually not very
definite. The rather rare case of a more or less definite sectorial
arrangement is illustrated in PI. IX, fig. 2. More coiiiinonly there is a
tendency for cells of one kind, either green or yellow, to be distributed
about the median line of the leaf the peripheral parts consisting of cells
I if the other kind; the boundary between the two parts is nearly always
irregulai'. A plant in which the green tissue occupies the middle of the
leaf is shewn in PI. IX, fig. 1. In such a plant, yellow cells constitute
the whole of the jjeripheral parts of the leaf, and very often the
sub-epidermal layer in the green region is also yellow, only the
internal layers being green. The plants are not, however, true periclinal
chimaeras; the yellow cells are not confined to the peripheral layers,
but occur also in other layers and may be scattered sporadically among
the green cells, while, conversely, isolated groups of green cells may
occur among the yellow cells. As is the case in most plants, the cells
of the epidermis (other than the guard cells of the stomata) contain
only colourless plastids, even in the normal green leaves ; one curious
exception to this rule has, however, been found. The case was that of

R P. Gregory 309

a variegated plant, in which the central parts of the leaves were green.
In one very young leaf, besides the central patch of green, there was a
faint green stripe along the margin of the leaf, and in this region it was
found that the epidermal cells alone contained definitely coloured
plastids (PL X, fig. 9), those of the deeper layers being nearly colourless,
as they usually are in the very young stages of the yellow tissues.

In most of the variegated plants there occur patches which are
intermediate in colour between the full gi-een and the clear yellow
(PL IX, figs. 1 — 3). Gradations of this kind are due to the presence of
more or fewer layers of normal green cells in particular regions. In one
variegated plant the mosaic consisted entirely of patches of full green
and lighter green ; that is to say, green cells were present in some
layers, at least, in all parts of the plant.

In the variegated plants, the stems, sepals and other organs, the
cells of which contain chloroplasts, have a structure similar to that of
the leaves.

In the pure yellow-leaved plants (i.e. plants which have no normal
green plastids at all), the very young stems and leaves are always of a
pale yellowish-white colour. The rate of growth of these plants is very
slow as compared with that of the variegated plants, and still more so
as compared with that of the pure green plants (see PL IX, fig. 3, in which
three sister plants of the same age ai-e shewn). Most of the yellow-
leaved plants die at an early stage ; in such as survive, the pigmentation
of the plastids increases somewhat in the older leaves, which become
yellowish-gTeen. Apart fi'om this change with age, various yellow-
leaved plants may also shew some slight gi-adation, from a less to a
more pronounced greenish tint, which is no doubt due to different
degi-ees of chlorosis of the constituent cells. In some ca.ses, plants,
which have no normal green cells, have, side by side, cells of different
degrees of chlorosis (PL X, fig. 8) ; thus the plant is built up of a patch-
work of different kinds of cells, and in that respect is comparable witii
a variegated plant, although it is, nevertheless, chlorotic throughout.

So far as the fully gi'own organs of the variegated plants are con-
cerned, no exception has been found to the rule that any particular cell
contains chloroplasts of one kind only, though there may be minor
variations in the size of the individual chloroplasts. But in the very
young, actively growing leaves, evidence has been obtained of the
existence, side by side in the same cell, of chloroplasts of different
kinds, which differ from one another in the same way as do the chloro-
plasts of the normal and of the yellow cells in the mature organs.

310 Variegation in Primula sinensis

Cells containing different kinds of plastids have been foinul to occur
along the lines of junction between gi'oups of green and yellow cells.
In such cells, as seen in sections taken from fresh material, the differ-
ences are readily recognizable, not only in the size, but also in the
colour and starch-content, of the chloroplasts. Fig. 10, PI. X, is from a
fresh preparation. In this case, the cells 6', D. E and F contained only
chlorotic plastids, but each of the cells A and B contained both large
bright-green chloroplasts of the normal type and also smaller pale-
coloured plastids (c, c, c), indistinguishable in appearance from the
pale-coloured plastids of the chltinitic cells. The chlorotic plastids of
the cells C, D, E and F were not all exactly alike, the majority of those
contained in the cell C being definitely of a pale yellowish-green colour,
while most of those contained in the cell D were nearly or quite colour-
less; in each cell, however, both kinds of chloroplast were represented,
C containing a few colourless plastids (d, d, d), T) containing a few pale-
coloured plastids (c, c, c). The j^lastids of E were jjale-coloured, those
of F colourless. The difference between these two kinds of chlorotic
plastid is, of course, by no means so sharp as the difference between
the chlorotic and the normal plastids; too much stress should not be
laid upon it, but it suggests that the plastids of the cells C and D
represent a mixture of two kinds of chlorotic plastids, analogous to the
mixture of normal and chlorotic plastids found in A and B.

The use of fresh material for observations ol' the kind just described
is open to certain objections, chiefly on account of the risk of confusion
due to the displacement of chloroplasts from the cells to which they
rightly belong. The observations have, therefore, been checked by the
examination of fixed material, cut in parafBn and stained. By the use
of this method under suitable precautions the risk of error due to dis-
placement of the chloroplasts can be almost eliminated, and systematic
searching is much facilitated. The method suffers from the disadvantage
that differences of colour between the chloroplasts are no longer recog-
nizable and the only distinction is one of size. Even in the very young
leaves examined, the enormous majority of the cells contain only one
kind of plastid, either large or small, but a certain number of cells have
been found, always near the boundary between patches of normal and
chlorotic tissue, in which there is no doubt that chloroplasts of different
sizes exist side by side. Some of these cells are illustrated in PI. X,
figs. 11—18.

In view of these observations, and their probable significance in
relation to the maternal inheritance of variegation, it is desirable that

R. P. Gregory

311

investigations should be made, with the object of tracing back these
differences between the plastids to the earliest stage in development at
which they can be recognized. At the present time no material is
available for this purpose, but I hope some may be obtained next
year.

Breeding Experiments.
The original variegated plant, from which my variegated race has
been bred, appeared in the F., from a cross between the two normal
green races Ivy-leaf and Snowdrifts The F^ from this mating consisted
of normal green plants, two of which were selfed ; one of them gave a
large family, consisting of 160 plants, one of which was variegated.
The variegated plant was selfed and gave a family containing green,
variegated and yellow plants. Two of these variegated plants again
were selfed, and gave families containing the same three classes of
offspring. The foregoing results are set out in detail in Table I.

TABLE I.

Ivy-leaf x Snowdrift

I
F, 45/09

13 plants, all green

451

452

59/10
129 plants, all green

93/11
38 plants, all green

I

I
99/12

GO/10

159 green 1 variegated

I I

602 601

92/11

25 green ti variegated 7 yellow

92' 92-' 92" 92''*

I
I

100/12

48 gi-een 12 variegated 10 yellow

993

251 green 16 variegated 38 yellow
1003-8*

1001

100- «

991 99-

The plants were selfed but in each
case the inflorescence died

* Used in crosses ; see Tables II and III.

' For a description of these races see Journ. of Genetics, Vol. i. p. 102, 1911.

312 Variegation in Primula sinensis

There is one further point in connexion with these experiments which
should be mentioned, namely, the fact that the flaked type of flower-
coloration was contributed to the original mating by the Ivy-leaf
parent, and this character appeared in each of the subsequent gene-
rations. In families containing green, variegated and yellow plants,
both self-coloured and flaked flowers occurred in each of the three
classes of offspring. The flaked character of the flowers is seen, there-
fore, to be quite independent of the variegation of the leaves, and
it is, in fact, a character of a different nature from that of variegation,
for it is inherited through the male, as well as through the female,
gamete'.

The extracted variegated and yellow-leaved plants have been used
both as male and as female parents, in crosses with normal green
plants. The results of these experiments are shewn in Tables II and III.
It will be seen that when the yellow-leaved plant is used as the male
parent, both the Fi and the succeeding generations consist entirely of
normal green plants ; neither variegated nor yellow-leaved j^lants appear
among the progeny of these matings. When, on the other hand, the
yellow-leaved plant is used as the female parent, the Fi is yellow.
Owing to the slow growth of the yellow-leaved plants, and the con-
sequent lapse of time before they bear seed, I have only just obtained
the seedling i^i-plants from crosses in which the yellow-leaved plants
were the female parents ; but there can be no doubt, I think, that the
yellow-leaved plants can only give yellows in succeeding generations.
It will be noticed that matings between green and yellow, whichever
way they are made, do not lead to the production of variegated plants ;
the progeny are all either self-coloured green or self-coloured yellow,
according to the character of the mother.

For the sake of simplicity, I have omitted from the Tables any
reference to characters other than those of green, variegated and yellow
leaves. Each of the F^'s was, however, heterozygous in respect of a
series of factors, some of which were derived from the male, some from
the female parent. All these factors underwent normal segregation in
the F.Js shewn in Table II. This result is quite in accordance with
anticipation and the only point upon which it is necessary to remark is
the fact that the flaked coloration of the flowers, observed in the families
106/18 and 107/13, was derived from the male parent used in the flrst
cross.

' See also Correns, Zeitschr.f. Intl. Abst.- u. Vercrhungslehre, i. p. 322, 1909.

R. P. Gregory

313

TABLE II.

Matings of Normal Green 9 '■'• Variegated or Yelloiv-haved $ .

Mating Fi

Rosy-magenta* X 92711 28/12 24 plants.
Normal Green Yellow- All Green

leaved

ditto X ditto 29/12 17 plants.

All Green

ditto X 92«/ll 30/12 2 plants.

i'ellow- All Green

leaved

Crimson King* x 92"/ll 159/12 27 plants.

Normal Green Yellow- All Green

leaved

11/13 78 plants. All
Green; no Varie-
gated; no Yellow

12/13 (not sown)

j 106/13 114 plants.
All Green

1 107/13 85 plants.
I All Green

( 101/14 28 plants.

j All Green

-J 162/14 40 plants.

All Green

163/14 5 plants.
All Green

* For a description of these races, see Journal of Genetics, Vol. i. p. 102.

TABLE III.

Matings of Variegated or Yellow-leaved 9 x Normal Green $ .

Mating
100/12 X
Variegated

Number of flowers
pollinated

4/12 5
Normal Green

No seeds

100/12
Variegated

X

ditto

4

No seeds

100/12
Yellow-leaved

X

Crimson King
Normal Green

7

21714

5 plants. All Yellow

100/12
Y'ellow-leaved

X

ditto

2

41714

5 plants. All Yellow

100/12
Yellow-leaved

X

ditto

2

33714

1 ijlant. Yellow

100/12
Yellow-leaved

X

32/12
Normal Green

2

No seeds

100/12
Yellow-leaved

X

ditto

1

No seeds

Discussion of an hypothesis.

1*116 work of Correns on Mirahilis^ ha,s given results of particular
interest, because of the relations which the form albomacidata has been
shewn to po.ssess, not only with the normal green type, but also with
the chlorina races. The form chlorina is distinguished from the normal
green type by the pale green colour of its leaves, which is due to the
relatively small amount of pigment developed in the chloroplasts. In
crosses between the normal gi-een and the chlorina form, the normal is
dominant to the chlorina character, and segregation takes place in the

1 Zeitschr. f. Ind. Abst.- u. Vererbungslehre, i. p. 291, 1909; and 11. p. 331, 1909.

314 Varieqathn In Primula sinensis

usual way. When the cidorina $ is crossed with albumuculata ^^ , the
hybrids so formed are indistinguishable from hybrids between the
chlorina and the normal green races, both in appearance and in the
progeny to which they give rise. That is to say, the pollen grains of
the albomaculata race, even when they are produced by flowers borne
on the white branches, nevertheless caiTy the fiictor for normal
green, as distinguished from the pale green of the chlorina. In these
races there are, therefore, two distinct characters of the chloroplasts,
one of which is inherited through the mother only, while the other is
inherited in the usual maimer. Correns suggests' that these facts may
be explained by means of an hypothesis based on the assumption, which
has received some degree of support from cytological observations, that
in the process of fertilization in the higher plants the nucleus of the
male gamete passes over to the egg-cell alone and unaccompanied by
any cytoplasm, so that the cytoplasm of the zygote is entirely of
maternal origin. The germ-cells of the albomaculata race are regarded
as possessing nuclei which are perfectly normal and carry the factor for
the typical green leaf-colour ; the cytoplasm of the germ-cells, however,
in correspondence with the mosaic of which the plant consists, is either
normal or chlorotic ("gesund oder chlorotischkrank ") and, accordingly,
either permits or prevents the development of normal gi-een chloro-
plasts. Assuming the cytoplasm of the zygote to be entirely of maternal
origin, the offspring of a variegated albomaculata plant are, therefore,
green, variegated or yellow, according as the cytoplasm of the egg-cells
is entirely normal, a mosaic of normal and chlorotic components, or
entirely chlorotic.

In formulating this hypothesis, Correns'' leaves it an open question
whether the seat of the abnormality in the chlorotic plastids is to be
looked for in the plastids themselves, or in the cytoplasm which surrounds
them. Baur, in discussing his experiments on Pelargonium", inclines to
the former alternative ; Correns regards the latter as at least equally
possible in Mirabilis.

In Primula no form is yet kno^vn corresponding with the chlorina
races of Mirabilis, but the maternal inheritance of the variegated and
yellow-leaved characters in Primula corresponds exactly with that of
the albomaculata character in Mirabilis and is, I think, to be explained
on lines similar to those put forward by Correns. But the evidence

' Zeitschr. f. Ind. Abst.- u. Vererbunr/slehre, ii. p. 331, 1909.

= L.C. p. 332, footnote (2).

' Zeitschr. f. Ind. Abst.- ii. Vererbvngslfhi;; i. pp. 348 ff., 1909.

R P. Gregory 315

which I have obtained, in the very young leaves of variegated Primulas,
of the existence of normal and chlorotic chloroplasts side by side in the
same coll, appears to me to afford definite support for the view that the
abnormality is localized in the chloroplasts themselves, and is not a
function of the cytoplasm as a whole.

In the present state of our knowledge of the functions of cytoplasm
and nucleus, the question is one of some importance. If the maternally
inherited character pertains to the cytoplasm in general, as contrasted
with the nucleus, the consequence is, as Correns has remarked, strongly
to emphasise the importance of the part played by the nucleus, at the
expense of any part the cytoplasm might be supposed to play, in the
transmission of characters which are inherited through the male and
female equally. The hypothesis that this function is limited to the
nucleus is, on diverse grounds, regarded favourably by many cytologists,
but there are still difficulties to be solved before its complete acceptance
can pass unquestioned. Moi-eover, for the present purpose, no assump-
tion in this respect need be made, if the view be justified that the
abnormality of the chloroplast, which is inherited through the egg-cell
only, is localized in the chloroj)last itself. Such a view permits of a
modification of Correns' hypothesis, which would serve to account for
the' maternal inheritance of the character with which we are dealing,
while leaving untouched the question as to the relative functions of
nucleus and cytoplasm (apart from the chloroplasts) in the transmission
of characters which are inherited in the usual way.

Since the work of Schimper' and others thirty years ago, the view
has gained general support and acceptance that the jDlastids of a plant-
cell are persistent cell-organs, in the sense that they are invariably
formed by the division of previously existing plastids and are handed
down from mother to daughter cell. It is, then, reasonable to suppose
that an abnormality inherent in the plastid itself would be handed on
to the products of its division^, so that abnormal plastids would give

' Jahrb.f. wissensch. Bot. xvi. pp. 1 — 247, 1885. Keferences to recent literature con-
nected with this subject are given by Cavers, Neic Phytologist, xiii. pp. 96 — 106 and
170—180, 1914.

- If it be granted that the abnormality, with which we are deahng, is inherent in the
chloroplast itself, the genetics of the normal, variegated and chlorotic plants provide
what amounts to a proof of this proposition. The original variegated plant, which was
the offspring of pure green parents, is an exceptional case. But the rarity of such
exceptions — only one has occurred among many thousands of plants — and the fact that
the partially or completely chlorotic progeny of that plant has continued invariably to
throw plants with chlorotic plastids, is additional support for the view that normally the
products of the division of a plastid are like the plastid to which they owed their origin.

316 Variegation in Primula sinensis

rise by division lo further abnoniial ones, while the products of the
division of a normal plastid would be normal. In order to explain the
maternal inheritance of this abnormality, it is necessary to adopt
the hypothesis, which has met with general acceptance as an cxtcnsiffn
of tihe general theoiy of the persistence of plastids, that the plastids of
the zygote are derived solely from those present in the unfertilized egg.
Thus we should reach an explanaticjn of the fact that thi^ progeny of
pure-green normal and pure-yellow chlorotic plants are, respectively, all
green and all yellow, no matter what was the character of the male
parent. The variegated plants are invariably the offspring ot \aiiegated
mothers and they give rise to green, variegated and yellow offspring.
The green and the yellow offspring may be explained as originating
from egg-cells formed in the pure green or pure yellow patches of tissue
which occur in the variegated mother-plant. The variegated offspring
must be supposed to have their origin from egg-cells, which are endowed
at their formation with a mixture of plastids of different kinds, such as
has been found in the cells of the young leaves of the variegated
Primulas. The segmentation of a fertilized egg containing different
kinds of plastids, each giving rise by its division to plastids of its own
kind, would tend to a gradual soi'ting out of the different kinds of
plastids into different daughter cells. Eventually, as Baur has pointed
out', the great majority of the cells would contain plastids of one or
other kind only, and the adult tissues would be a mosaic of cells, the
pattern of which would depend upon the distribution in the embryonic
cells of the different kinds of plastids.

The foregoing hypothesis rests primarily upon the general theory of
the persistence of plastids from cell to cell in a series of cell-divisions,
and the extension of that theory to the effect that the plastids of the
adult are genetically derived from those present in the egg. Both the
theory and its extension have formed the subjects of an extensive
literature with which it is impossible to deal in detail here. Suffice it
to say, that the theory of the persistence of the plastids has met with
very general support and acceptance for many years. The question has,
in some respects, been re-opened by recent investigations on chon-
driosomes in plants", but the work which has been done in this direction
has not as yet yielded any well-established results ; such as they are,

1 L.C. p. 349.

" A general review of this work has recently been given by Cavers, New I'ln/toluyixt,
XIII. pp. 96—106 and 170 — 180, 1914. I am greatly indebted to Dr Cavers for his kindness
in giving me an advance proof of the second part of his article.

R. P. Gregory 317

they tend, I think, not so much to call in question that aspect of the
theory with which we are concerned, as to support the view that the
plastid-origins arc handed on from parent to offspring as definite
bodies.

Granting that the plastids are persistent cell-organs, the existence
of mixtures tif plastids of different kinds in certain cells renders it
difficult to resist the inference that the abnormality lies in the plastid
itself and not in the surrounding cytoplasm. The inference is supported,
I think, by the fact that different degrees of chlorosis may occur in the
same plant, and by the fact that some entirely chlorotic plants have
been found to be mosaics of cells, the plastids of which exhibit well-
marked differences in the degree of chlorosis.

The hypothesis that, in the higher plants, the plastids of the s^ygotc
are genetically derived fi-om those present in the unfertilized egg-cell
has, like the general theory of which it forms an extension, been widely
accepted ; but it is obvious that it remains an assumption for any
particular species, until that species has been the subject of special
investigation. In any case, however, the assumption that no plastids
(or plastid-origins) pass over in fertilization, from the male to the
female gamete, demands less than does the alternative assumption that
no cytoplasmic structures of any kind accompany the male nucleus into
the egg-cell. In many cases the nucleus of the male cell has been
described as becoming disengaged from the cytoplasm, which does not
enter the egg-cell ; on the other hand, there are cases among the
higher plants, in which the male generative cell as a whole enters
the egg, and, in some cases, bodies resembling leucoplasts have been
observed in the mass of cytoplasm brought into the egg with the male
cell'. This may, however, be compared with the behaviour of the
chloroplasts in certain species of the alga Spirogyra. In this genus,
the two gametes contribute almost equally to the cytoplasm of the
zygote, the chloroplast (oi- chloroplasts) of the male gamete passing
into the zygote along with the other structures of the male cell ; but
Chmielewsky- and Trondle^ have shewn that the chloroplast of the
male gamete degenerates after entering the zygote, while that of
the female gamete alone persists and becomes the chloroplast of
the zygote.

" For instance in Pinus. See V. H. Blackman, Phil. Trans. Roy. Soc. B. Vol. 190,
1898, pp. 395—426.

2 Bot. Zeit., Jahrg. xlviii. pp. 773—789, 1890.

» IM. Zeit. Jahrg. lxv. pp. 187—216, 1907; Zeitschr.f. Hot. iii. pp. .593—019, 1911.

318 Variegation in I'rinmla sinensis

In the present connexion, the i-ecunt investigations on chondriosomes
in plants, to which reference has already been made, rexjuire some con-
sideration, which, however, need only be of the briefest, as the subject
has recently been dealt with fully in a general review by Cavers'. The
chondriosomes are bodies, the presence of which in the cytoplasm of
many organisms has been demonstrated by means of appropriate methods
of fixation and staining, 'fhey have been described as persistent cell-
organs and are regarded by some writers as homologous with the
mitochondria of animal cells, to which Meves and others have attributed
important functions in the determination of heritable characters. In
another direction, the suggestion has also been made that the plastids
of plants are derived from the chondriosomes present in the embryonic
cells. At the present time, however, our knowledge of chondriosomes
has by no means reached the stage at which any definite conclusions
can be drawn as to their sigiuficance. The real nature of the bodies
which have been described under this name is still a matter of con-
siderable uncertainty'; whether they are persistent cell-organs, and
whether the chondriosomes of plants are homologous with those of
animals, still remains to be proved, while the suggested relations be-
tween the chondriosomes of plants and plastids are very much open to
(luestionl Moreover, apart from the uncertainty attaching to the fore-
going points, there is, so far as I know, no definite evidence of the
transference of chondriosomes from the male generative cell to the egg-
cell, in the fertilization of higher plants, even in those cases in which
chondriosomes have been described as occurring in the developing
gametes of both sexes.

So far, then, the study of cliDndriosomes has not afforded any results
sufficiently well established ti> be adduced either in support of, or in
opposition to, the hyf)othesis which has been put forward in explanation
of the maternal inheritance of certain forms of \ariegation and chlorosis.
It is much to be desired that investigations should be made on variegated
plants, by means of the improved methods employed in the stud}- of
chondriosomes, with the object of tracing back the ditt'erences between
the plastids, which, have been observed in the cells of the young leaf, to
the earliest stages in development at which they can be recognized. It

' '■ Chondriosomes (Mitochondria) and their significance,'" Ncio Phijtolotiist, xiii. pp.
'Jfi— 106 and 170—180, 1914.

- See Cavers, I.e. p. 175, on the resemblance of chondriosomes to myeUn forms.

■• See, for instance, the recent paper of Scherrer, "Untersuchungen iiber Ban und
Vermehrung der Chromatophoren und das Vorkommen von Chondriosomen bei Antho-
ceros," Flora, N. F. lxx. pp. 1— 50, 1914.

R. P. Gregory 319

may be hoped that such investigations will lead to opportunitfes for
observing the distribution of plastids to the daughter cells, during the
division of cells containing mixtures of plastids of different kinds. In-
vestigations should also be made into the development and structure of
the egg-cells formed in the green, variegated and yellow parts of the
plant, and into the process of fertilization.

In the hypothesis which has been put forward above, the suggestion
has been made that the character, which is inherited through the egg-
cell only, is inherent in the plastid itself and is therefore handed on to
the products of its division ; in other words, that the plastids are self-
determining in respect of the character under consideration. But if
this is so in regard to characters of a particular class, there are other
characters in which it is not the case. The chlorina character of
Mirabilis, and the plastid-colours of many flowers, are inherited through
both sexes equally and undergo normal segregation. Their inheritance
may be expressed in terms of Mendelian factors, and it may be presumed
that the means by which they are transmitted is the same as that of
other Mendelian characters, whatever that may be. The hypothesis of
Correns would assign to the nucleus the function of transmitting the
factor, which distinguishes the normal green from the chlorina races of
Mirabilis; at the expense, however, of making the assumption that the
nucleus alone passes over from the male cell to the egg-cell in fertiliza-
tion. The modification of the hypothesis indicated above would suggest
that this assumption is unnecessary, in order to explain the jjurely
maternal transmission of certain chai'acters of the chlorojjlast, such as
those with which we are dealing. On the other hand, it leaves un-
touched the question as to the relative parts played by nucleus and
cytoplasm in the transmission of ordinary Mendelian characters, among
which certain other characters of the plastids are included.

Part of the expenses of the experiments described in this paper was
defrayed by means of grants from the Royal Society and from the
British Association. A large number of plants were grown for me at
the John Innes Horticultural Institution and I desire to express my
great gratitude to the authorities for the facilities which they have
continued to extend to me.

•Journ. of Gen. iv 21

320 Variegation in Primula sineiihiis

DESCRIPTION OF PLATES.

PLATE IX.

Fig. 1. A variegated plant with the green tis.siie nccu|iying the central regions of the

leaves, the yellow tissue occupying the peripheral part.
Fig. 2. A variegated plant shewing a sectorial arrangement of the pure yellow tissue.
Fig. 3. Three sister plants from the family 99/12. The plants were of the same age and

were grown together under the same conditions. They illustrate the difference in the

i-ate of growth between the pure green (on the left), the variegated (on the right) and

the pure yellow-leaved (above) plants.

In the foregoing figures, the arrows point to lines of junction between full green and
paler green areas. This difference in colour is due to the presence of more layers of
normal green cells in the full green areas than are present in the areas of a lighter green
colour.

PLATE X.

Figs. 1 — 8 and 10 are from sections of fresh material. Figs. 9 and 11 — 18 are
from .stained preparations. All the figures were drawn with the aid of a camera lucida.

Fig. 1. Chloroplasts from the palisade cells of the leaf of a normal green plant, after

exposure to bright sunlight, shewing the starch grains. x 650.
Fig. 2. Chloroplasts from the palisade cells of a yellow-leaved plant, similarly treated.

The plastids are much smaller and contain much less starch, x 650.

Fig. 3. A palisade cell from the leaf of a normal green plant, shewing the chloroplasts
after the starch had been removed by keeping the plant dark for some days, x 440.
Fig. 4. A cell from the mesophyll of the same plant, x 440.

Fig. .5. A palisade cell from the leaf of a chlorotic plant for comparison with tig. 3.
x440.

Fig. 6. A cell from the mesophyll of the same chlorotic plant, x 440.

Fig. 7. Cells from the mesophyll of the leaf of a variegated plant after exposure to sun-
light. Two of the cells shewn contain normal green chloroplasts ; the others contain
plastids exhibiting various degrees of chlorosis, x 440.

Fig. 8. Palisade cells from a pure yellow-leaved plant. This yellow-leaved plant con-
sisted of a mosaic of cells of different degrees of chlorosis, and in that respect was
comparable with a variegated plaut, though it was, nevertheless, quite without normal
chloroplasts. Of the three cells shewn, the middle one contained plastids exhibiting
an extreme degree of chlorosis; in the plastids of the other two cells the chlorosis
was of a less extreme type, x 650.

Fig. 9. Section through a very young variegated leaf, in a region in which the epidermis
alone contained coloured chloroplasts. The well-developed plastids of the epidermal
cells are shewn ; all the other cells in this region of the leaf contained only small,
chlorotie plastids (see p. 309). x 650.

R P. GrRECxORY 321

Fig. 10. Section of a very young variegated leaf, examined in the fresli state. The
section represents a group of cells at the junction between a patch of green tissue and
the surrounding chlorotic tissue. The cells A and B contained (1) bright green
chloroplasts of the normal type, with well-developed starch-grains, and also (2) smaller
plastids, c, c, c, of a pale colour and with little starch, which were indistinguishable
from the pale-coloured plastids of the cell C. In most of the plastids of the cell C
the pigment was readily recognizable, but in a few, d, d, d, no colour could be detected
with certainty. The plastids of the cell D were, for the most part, nearly colourless
in appearance, but three or four, c, «, c, could pass for the pale-coloured kind found
in C. The cells C and D, therefore, contained plastids differing from one another in
the degree of chlorosis. E and F were also chlorotic cells, as were all the cells on
this side of the section. The plastids of E were of the pale-coloured type ; in none of
those of F could any colour be detected with certainty, x 650.

Figs. 11 — 18 are drawn from very young variegated leaves, examined in fixed and
stained preparations. The material was cut in hard paraffin, so as to avoid, as far as
possible, the risk of crushing the cells and consequently displacing the chloroplasts from
the cells to which they properly belonged. No indication was obtained that any dis-
placement had taken place, and all cells, the walls of which were ruptured, were rejected.
The sections were cut so as to pass through adjacent patches of normal and chlorotic
tissue. The stains employed were Carbolic Fuchsin and Light Green. This method did
not give any definite differential coloration as between the normal and chlorotic plastids,
BO that it was necessary to rely upon differences in size as the distinguishing feature. The
figures represent cells containing chloroplasts of different sizes ; all these cells were found
at the junction between the normal and the chlorotic tissues. In certain cases, where two
smaller chloroplasts lie close together in a pair, it was not always possible, with the method
of staining employed, to say whether they were chlorotic plastids, or whether they were
the products of a recent division of a normal plastid ; but it is clear that the possibility
of accounting for some of the small plastids in this way only applies in the minority
of cases. This sort of difficulty is much less in preparations of fresh material, where the
differences in colour form an additional guide. Figs. 11 — 18 are all x 650.
Fig. 11. The chloroplast, x, is a large one seen in end view. Near it is another large
one and also a small one, both seen in face view. The two smaller chloroplasts to
the left of the cell may be the products of the recent division of a normal one.
Fig. 12. Cell containing two large chloroplasts and several small ones. The two chloro-
plasts at (/) were in different planes, near the upper and the lower walls of the cell
respectively.
Fig. 13. The palisade cells (above) contain only small chloroplasts. Below, to the right,
are three cells containing large chloroplasts, some of which are seen sideways or
end-on. To the left are two cells each containing both large and small chloroplasts.
Fig. 14. Cell containing several large and one small chloroplast. All the chloroplasts

contained in this cell are shewn in the figure.
Fig. 15. The four chloroplasts shewn were in focus together.
Fig. 16. Cell containing four large and five small chloroplasts.
Fig. 17. Cell with three large chloroplasts and several small ones.

Fig. 18. The cell shewn in the middle had chloroplasts of different sizes ; those above had
chlorotic plastids, those below large ones only.

21—2

JOURNAL OF GENETICS, VOL. IV. NO. 4

PLATE IX

i'lg. 1.

Fig.

i'ig. 3.

JOURNAL OF GENETICS, VOL. IV. NO. 4

PLATE X

0

t^ d)

8

A SECOND BRACHYDACTYLOUS FAMILY.
By H. DRINKWATER, M.D., F.R.S. (Edin.), F.L.S.

I HAVE already published accounts of three families showing an
inherited symmetrical shortness of the fingers and toes in individuals
who are also below the average in stature. In one — the " Brachy-
dactylous Family'" — the fingers are reduced to about half the normal
length : in the other two families they are intermediate in length
between these very shoi-t ones and the fingers of average normal
individuals, a condition which I have designated by the term "Minor-
Brachydactyly^"

In both types, the shortening was shown to be due chiefly to an
abortive condition of the middle phalanx. The main features are
represented in the outline illustrations in Fig. 1, where A shows the
bones of the middle finger of a normal adult, C the Brachydactylous
condition, and B the Minor-Brachydactylous.

In G the middle phalanx (2) is seen to have become ankylosed to
the terminal phalanx (3).

In the Summer of 1913, my friend Dr J. D. Lloyd informed me that
he knew of some people resident in his neighbourhood whose hands
closely resembled those of one or other of the above-mentioned families;
and he not only afforded me an opportunity of examining some of them
at his own surgery, but very generously consented to allow me to cany
out whatever investigations I might be disposed to undertake with
regard to them.

Examination at once made it evident that the abnormalities in
these new cases are identical with those described in my paper on
" Braehydactyly " ; there is, namely, the maximum amount of shortening
of the digits, and the same characteristic shortness of stature. The
essential peculiarities are so exactly similar that one cannot resist the

' "An account of a Brachydactylous Family," Proc. Roy. Soc. Edin. Vol. xxviii. Part i.
" "Account of a Family showing Minor-Brachydactyly," Journal of Genetics, February,
1912. " Minor-Brachydaetyly " No. 2, Journal of Genetics, February, 1914.

324

A Second Brachydactjjlons Family

conviction that the two families must have descended from the same
original stock.

The connection between them unfortimatejy cannot now be
established, for there is not a single surname common to the two
families. This difference in names may be accounted for by the

ABC

Fig. 1. Normal and Biachjdactylous Phalanges. (Natural size.)
A. Normal. B. IWinor-Brachydactylous. C. Bracbjdactylous.

fact that the inheritance in the second famil}- has been almost ex-
clusively through the female line, so that the suinaiin- has changed
(through mari'iage) at each generation.

Both families reside in the same part of the country: a fact which
would seem to give some support to the theory of their common origin.

Dr W. C. Faraboe was the first to describe the condition of Brachy-
dactyly in a fiimily that he studied in 190.S' in North America. Is

' Published in March 1905 by tlie Peabody Institute, Harvard, U.S.A.

H. Drinkwater 325

there any blood relationship between this American family and either
of the English families ? The cases observed by Farabee were ap-
parently identical as regards the anatomical and hereditary features
with those described in my first paper in August 1907. I was not
aware of Farabue's work until my own investigations were nearly
completed, and there was not time to decide the question of relation-
ship before presenting my paper to the Royal Society of Edinburgh.
His reply to my enquiries did not come for several months afterwards,
for he happened to be in some out-of-the-way part of Peru engaged on
a scientific expedition. However, it was found that no surname was
common to the two families ; nor was it possible to make my chart fit
in with his : so that, assuming that there had been a common ancestor
of the Brachydactylous type (and this hardly admits of doubt), he or
she must have lived pi'ior to the earliest member now traceable in
either of the two families. A study of Farabee's account made me
think it likely that his family had descended from "an abnormal
member of the English family, four generations back, who had migrated
to America, but of whom no tidings have since been received by his
relatives in this country," but as this man's name does not occur in
Farabee's family, he was obviously not the connecting link. So far,
therefore, I have not been able to prove any blood relationship as
existing either between my two families or between the first of these
and Farabee's.

There still remains the question whether the second family (about
to be described in the present communication) is related to Farabee's.
Farabee informed me that the name amongst his people which most
nearly approached any name in the English family was Hyde. Now
the man marked 2 in the chart on page 327 was named Benjamin
Hyde. In November of 1913 when the chart was almost complete,
I forwarded Dr Farabee a copy of the earlier generations, numbering
each individual 1, 2, 3 etc. My letter was a long time in reaching him,
for again he was away from home on another scientific expedition — this
time in Brazil. I asked if there was a Benjamin Hyde in the American
family ? At the end of May I received a letter from him from Barbados,
dated May 16th, 1914, in which he says:

" Your letters of November 26th, 1913, and February 9th, 1914, have just reached
me. For the past ten mouths I have been in the interior of Northern Brazil and
Southern British Guiana, out of touch with the rest of the world.... FoM haee
settled the iphole question. 2 is Benjamin Hyde. His mother had short fingers, and
she was the only one of her family who had. She had eleven children, but I was

326 A Second BracJii/dactt/lons Famllji

uiial>le to learn even the sex of three of them marked ??? as tlioy died young.
1 had hoped to U)ok up the Enghsh branch of this family, but am glad to know
you have found it. You will have plea.-^ure in tlie comparative study...."

Now, as the name Benjamin Hyde is a vi^y i-arc cinnbinjition, tliei'e
can be little doubt that Farabee's family and this .second English family
are blood relations and belong to the .same stock; the only difficulty
with regard to it is the fact that in tiie English there is one generation
more than in the American family. This may be owing to thi' fact that
it is eleven years .since the American chart was constructed, and also
because maniage may not have occuiued at such an early age over there
as in this country, for in the English family many of the women have
married whilst still "in their teens."

Moreover the relative proportion of abnormal males and females is
exactly the same in these two families, viz. 61 per cent, of women in
the American family and 61 per cent, in the English family. This may
be a mere coincidence, but nevertheless it seems rather remarkable, and
differs from the proportions found in my first family, where there were
19 females to 25 males.

Ficr. 2 shows Farabee's chart, of the American family, and Fig. 3 the
English family.

I I I I I n \ I I I I
d* W" (/ c/ c? ? ? T ? ? ?

I 1 h 1 1 1 1 1 1 1 1 1

XX

1 — I — ^ — I 1 — I I — I — I — I — I

X. X

I I I I I I I I I I r I I I I I I I I I I I r I I I I I I

Fig. 2. Farabee's Chart.

It will be observed that the second generation in the English family
is incomplete compared with the second generation in Farabee's chart,
but there is no real discrepancy. It was Benjamin Hyde's sister, who
removed from America and settled in this country, who became the
ancestor of the English branch. My informants know that her maiden
name was Hyde, that she had a brother named Benjamin Hyde, and
that their mother had several other children, but do not know their type
or sex. As the rest of the family remained in America and lived in

H. Drinkwatbr

327

h

328 A Second Braclii/dacti/lons Famihi

Farabee's neighbourhood, he would bo liktdy to get a more complete
record than I could obtain, and hence the difference in the second
generation in the two charts is accounted for.

It will be observed that Farabee has here indicated the descendants
of abnormals only ; my chart includes the offspring of norinals us well
as ahnormals.

The rest of this paper i-efers exclusively to the English family which
I have recently studied.

The abnormality, wherever it exists at all, affects hotli hands and
both feet, as well as the stature. Both hands and both feet are always
affected symmetrically.

The chart includes 50 abnormal individuals, of whom .S4 are living.
Through the great kindness of these people I have been able to obtain
photographs of the right hand of 30 of them, and radiographs of the
hand and foot of 29, as well as a few full length portraits ; — a most
satisfactory record, when one considers their very natural reluctance to
do anything calculated to draw attention to them individually. In fact
there are only two adults whom I have failed to persuade to pay a visit
to the photographer and radiographer.

An Example of Mendelian Inhekitance.

Students of Mendelism will at once recognise that Brachydactyly as
illustrated by this family conforms in a remarkable degree with certain
laws enunciated by Uregor Mendel. One of these laws states that a
' dominant" character is transmitted only by a member showing that
character, and not by a member showing the " recessive " character,
and that the recessives always breed true to the recessive character.

In this family the normal individuals are "recessives," and according
to the theory cannot bear abnormal (short-fingered) children; and the
chart shows that the normals have had normal children only. In other
words, every abnormal child has an abnormal parent. As there has
been no inter-uiarrying oi abnormals, it follows that each abnormal
child has had one abnormal and one normal parent — one showing
Brachydactyly, and one with the normal type of hand and foot.

When abnormal individuals — the offspring of parents of two distinct
types — marry normals, the offspring (in conformity with Mendel's Law)
should be of both types, in appi'o.ximately equal numbers — 50 per cent,
of normals and 50 per cent, of abnormals. That is what Mendel found

H. Drinkwater 329

in the case of two types of sweet-pea, and his i-esults have been
repeatedly confirmed by other observers. The normals (recessives)
should produce normals only : the abnormals (dominants) should pro-
duce normals and abnormals in about equal numbers. The chart at
once shows that the types produced accord with this theory. What
about the relative proportion of the two types among the children
of the abnormals ? The short-fingered children of abnormal parents
ought to be equal or very nearly equal in number to the normal-
fingered children of the same parents.

As a matter of fact this equality is not usually exact, and theory
does not require it to be so, for there is a chance element which comes
into play which makes this precise number uncertain and variable.
According to Mendel the germ cells of any individual, such as one of
these abnormals, are of two kinds. One kind of cell carries the factor
which can produce the abnormality, and is inherited from the abnormal
parent ; the other kind of cell, being inherited from the normal parent,
lacks this factor : and these two kinds of germ cells are present in the
individual in equal numbers. If this be true, it must of necessity
follow that the particular kind of germ cell which will take part in
any given fertilization, will depend, so far as we can tell, upion a chance
meeting ; just as there is a chance of drawing a black or a white marble
out of a hat which contains an equal number of each. It is a lottery
with equal chances for both kinds. Sometimes one kind will pre-
dominate, sometimes the other, but in the long run the numbers
will be apjjroximately, if not exactly, equal.

In this family there are 50 abnormals, so that according to Mendel's
theory there ought to be ahoid 50 normals. The actual number of
normals is 48.

Thus, instead of the exact 50 per cent, of abnormals which miglit
occur in strict accordance with theory, we have 51 '02 per cent. This
is sufficiently near to constitute this family a striking instance of
Mendelian inheritance in the human subject. This remark applies
also to the other instances of Brachydactyly published by Farabee and
mj'self, as well as to my illustrations of Minor-Brachydactyly.

If a normal member of this family were to ask whether if married
to another normal (relative or non-relative) there would be any risk
of having Brachydactylous children, one would be justified in replying
that there was no risk whatever of such children resulting from the
union, for, not in a single case has a short-fingered child been bom of
two normal parents. This abnormality thus differs in a remarkable

330 A Second Bracliydactyloxis Famil;/

manner fi'om certain liereditary diseases (such as colour-blindness and
haemophilia) which are known to appear in the male children of a
woman, who, though showing no sign of these diseases herself, is yet
able to transmit them from her affected male parent to her children.

There are six generations shown in the chart. All the individuals
of the first, second and third generations arc dead : of the abnormals of
the fourth generation, only two are still living (Nos. 8 and 9); of the
fifth and sixth generations, nearly every niembei- (of both fy[)es) is still
alive.

Of the 34 normals living at the present time, I am able to give
fairly complete details of 30, and very incomplete details of the re-
maining four.

I am greatly indebted to Dr Geoffrey Williams of Wre.xham, Dr John
of Stoke-on-Trent, and Drs Bythell and Boydell of Manchester for the
excellent radiographs, without which it would have been impossible to
arrive at a correct interpretation of the exact nature of this interesting
abnormality.

"The hands' and feet, as already stated, are abnormal in each
affected individual, and the feet are, if anything, more abnormal than
the hands, at least as regards their digits. The middle phalanx is
practically or virtually — though not actually — absent from each finger
and toe. The metacarpal bones are short and otherwise abnormal, but
the metatarsus is .scarcely, if at all, affected. Nor is the variation
limited to the hands and feet, for all the individuals, with the exception

of young children are below the average stature, as shown by a

reference to the table of measurements " (page 33<S). It will be well to
stud}' each feature in turn in the following order : hands, feet, stature,
etc., as measuriMl and as revealed by photography and radiography.

Facts revealed ky Photography.

A. Hands : External aspect.

Length. " The most conspicuous feature is the shortness, especially
of the fingers ; these are only slightly more than half the normal
length, sometimes even less than half, whilst the hand looks abnormally
broad. The middle finger, measured on the palmar surface from the

' All quotations such as this, where the source is not mentioned, are from my jiaper on
Brachydactyly read before the Royal Society (Edin.) and serve to show features common
to the two families.

H. Drinkwater 331

base or metacarpo-phalangeal crease to the tip, is normally as long
as the width of the palm at the knuckles ; in these people it is ap-
proximately half," the average length in the adults being |. " Sometimes
it is shorter than the first finger." The extreme shortness in one or
two cases is due chiefly to the metacarpal bone. The hand, measured
from the carpal end of the radius to the tip of the first finger, has an
average length of about 5 inches. The width of the hand exceeds the
normal in numerous instances.

Markings. " The skin creases are peculiar. Each finger shows
only one crease, corresponding to the space between the first and
third phalanx'. It i.s single, like the one present in ordinary fingers
opposite the second joint." The crease "opposite the first joint is
double in most normal hands."

" Whilst it would be difficult to tell thut there are only two
phalanges in each finger, from an inspection of its dorsal aspect, the
palmar view shows clearly that such is the case ; and this is confirmed
by radiography in the majority of instances."

" The palm shows two peculiar lines." " One runs straight across
the hand transversally. ..it appears to be a union of the palmist's lines
of " heart " and " head."

" The second line, often the deeper of the two, starts from the
middle of this transverse line and runs to the space between the first
and second fingers." This is shown very distinctly on Nos. 20, 38, and
39 (Plates XII and XIII). It is occasionally faintly visible in normal
hands, but as a rule is quite absent.

" The skin is loose, and the whole hand is soft and fiabby."

" The palm is very compressible, owing to the wide intermetacarpal
spaces." The fingers are all " double jointed," i.e. they can easily be
flexed dorsally by slight pressure applied directly to their palmar
surfaces.

" A slight lateral pressure makes the palm half an inch narrower,
without altering the plane of the metacarpals. The narrowing is not
due to a transverse folding of the hand."

" On flexing and extending a finger at the basal (metacarpo-pha-
langeal joint) slowly, one can feel that the opposing surfaces are not
uniformly curved as in the normal hand. At certain points the
phalanx seems to slide over a ridge. This is what one would anti-
cipate from an examination of the radiographs." " The ring-fiinger in
several instances is bent at the middle, so that the tip points towards

' A bone of the finger.

332 A Second BrachniJacfyloits Family

the middle finger." This inclination is sometimes so marked that, in
the position of rest, the ring-finger lies partly in front of the middle
one. Strangely enough the little finger tends to project away from
its neighbour, so as to leave a large gap between the two (PI. XII,
Nos. 27, 23, 41).

The skin of the back (dorsum) of the hand is very cc^arselj'
reticulated.

The mouth of the sweat glands is conspicuous.

The nails are well formed in every individual.

Strength of Grij}. It is considerably below the noiiuul average.

B. The Feet.

" Here the one main peculiarity is the shortness of the toes — each
one apparently having only two phalanges. They are also broad and
straight, with very little tendency to the extreme flexion of many
ordinary toes."

In the photograph of the plantar aspect of the foot of the child
(PL XII, No. 43), the toes are shown fully extended. Frequently the
length scarcely, if at all, exceeds the width, as in the three smaller toes
in this child.

Facts revealed by Radiography.

A. Hands.

The X-rays show that the middle phalanx is not really absent ; a
fact which could not possibly have been ascertained from the most
careful ocular and digital examination. Without the aid of radiography
one would conclude that there is an absence of the middle phalanx, and
even the X-rays might lead to the same conclusion if the hands of some
adults were the only ones examined. The young man, PI. XIV, No. 37, for
instance, has only two bones in each finger, and at first sight it looks
as though the middle bone — the second phalanx — is altogether missing,
whilst the other bones appear about normal; but if the terminal phalanx
is carefully compared with the corresponding bone in a normal hand it
will be seen that the base is much thicker than it should be. In most
of the abnormal fingers, this thickened base is seen to form almost
a perfect cube, being as broad as long. Whenever this condition is
present, it will be found that there is no vestige of the separate middle
phalanx.

H. Drinkwater 333

What is the nature of this cubical basal portion of the teiTninal
phalanx ? and what has become of the second phalanx ?

In the middle finger of every abnormal adult member of this family,
the second phalanx is always present as a separate bone, but invariably
it is so far reduced in length as to become a cube ; and the terminal
(third) phalanx is of the normal pyramidal shape. The ring-finger
also, in certain individuals, resembles the middle finger in these
particulars, having a normal-looking third phalanx, and a separate,
short, cubical middle j)halanx. Whenever the terminal phalanx has a
cubical base, this base corresponds in shaf)e with the second phalanx
present as a separate bone in the middle finger. In fact " this
cubical basal portion is the second (middle) phalanx that has become
ankylosed' to the terminal phalanx. The pyramidal distal portion of
these bones corresponds to the ungual phalanx and the basal cubical
portion to the middle phalanx."

" It does not exist as a separate bone in either the index or little
finger in a single adult."

" What, then, has happened to the middle phalanx ? It varies in
two respects from the normal :

1. It is always very short.

2. It generally becomes ankylosed to the base of the terminal

phalanx.

The fact that the middle phalanx is abortive, but not completely
absent, is proved conclusively by an examination of the radiogi-aphs
of the hands of young children, before ankylosis has occurred, and in
whom the middle phalanx is seen to be invariably present, but at the
same time abnormal. Each phalanx, in the ordinaiy (normal) hand,
consists at first of cartilage (gristle) and is gradually transformed into
bone, or, as it is termed, becomes ossified, such transformation being
limited for some time to two parts, one forming the great bulk or shaft
of the phalanx, and the other a thin plate or disc at the base. This
disc is termed the Epiphysis of the phalanx.

That narrow strip of the cartilage which is situated between the
bony shaft and the bony epiphysis usually remains unossified until
about the twentieth year in each phalanx. The epiphysis of each
phalanx is seen to be situated at its base ; but the epiphysis of
each metacarpal (palmar) bone is at the distal extremity. So long
as the strip of cartilage between the shaft and the epiphysis remains

^ Joined by bony union.

334 A Second Brachifdactylous Famihj

unossified, the bone can increase in length, but after its ossification
further growth does not occur.

The disc seen in some of the radiographs at the base of the terminal
phalanx looks very much like an epiphysis, but is really the second
phalanx in a very abortive condition.

In some instances in children there is only one piece of developing
bone visible between the shafts of the third and first phalanges ;
normally, three can be seen : namely, (1) the epiphysis of the third,
(2) the shaft of the second, and (3) the epiphysis of the second phalanx,
and it is only j)ossible by a comparison of several hands to say what
this single bone represents. I believe it always represents the shaft of
the second phalanx.

In no single instance does the radiograph show the presence of an
epiphysis at the base of the second phalanx.

It is thus clear that there is no real absence of the second phalanx in
any individual, but merely a rudimentary condition, and that at a certain
stage of development there is a union of this tuith the terminal phalanx.

" The essential feature of the abnormality apparently consists in an
absence of the epiphysis at the base of the second phalanx. It is
possible that the epiphysis is also missing, in some instances, from the
third phalanx, and that the two phalanges (second and third) consist at
first of a single piece of cartilage."

Premature union of shaft and epiphysis is of general occurrence in
this family. Such union should nut occur until about 20 or 21 years
of age. In No. 41, a girl of 15, it is already comjjlete, and the radio-
graph would pass for that of an adult hand. In this instance, moreover,
the union is not recent, for the epiphyseal lines are already almost
entirely obliterated. The hand of the boy (PI. XV, No. 31), aged 8 years
and 4 months, shows a very interesting peculiarity. The epiphysis of
the third phalanx, though still separate from its own shaft, has already
united with the shaft of the second phalanx in the first and little
fingers. This seems to point strongly to the opinion expressed above,
that these two phalanges are sometimes represented by only one piece
of cartilage. This premature union of the shaft and epiphysis is no
doubt one great factor iu, producing the characteristic shortening.

" The index and fourth fingers seem more aberrant than the second
and third, as they (the former) never show the middle phalanx as a
separate bone in the adult." In PI. XV, No. 27 (aet. 19), the union has
occurred in the little finger : in the index finger the union is not yet
complete, but it is evidently taking place.

H. Drinkwater 335

" Functionally, the fingers are all reduced to the bi-phalangeal
condition, and thus come to resemble thumbs."

The Thumb. " The change in the thumb consists of a shortening
of the fii-st phalanx which is reduced to a cube. There is no attempt
at ankylosis however." In some children the epiphysis is present, but
in others it is apparently absent, for if present union must have occurred
before the seventh year. The terminal phalanx has the epiphysis in
every individual.

" The so-called metacarpal bone of the thumb is in reality the first
phalanx, for here the epiphysis is clearly seen at the base, whereas in the
other metacarpals the epiphysis is seen at the distal end of the bones."

" The Metacarpals vary more or less in different individuals, but, as
a rule, where ossification is complete, they are abnormal." " The head
of each metacarpal is distinctly nodulated in many cases.... The middle
one is the shortest of the four," in No. 25, and in this woman the whole
palm shows the maximum degree of shortening. In PL XV, No. 27,
the metacarpals of the middle and little fingers are much shorter
than the other two.

"Sesamoid bones are well shown in several cases."

Spaces. The intermetacarpal spaces are generally increased in
width, especially between the heads of the bones. Sometimes it is
about three times the normal width. This accounts for the easy
compressibility oi the palm laterally.

Measurements : Average in Inches.

The following table gives the average length of the middle finger,
the hand, and the stature in eleven abnormal adult women (8 married
and 3 spinsters).

Middle Finger Hand Stature

1| 4-1 57i

In normal women these measurements are about 3, 6f and 64

respectively.

There is only one abnormal adult male member of the family now
living, and I have not yet seen him. The only measurement he has
sent me is that of the middle finger, which he says is 1§" in length.

There are three abnormal males between 17 and 21 years of age, and
their averages are as follows' :

Middle Finger Hand Stature

li 5| 61i

1 In the first family : these measurements were

1—15/16 54 61.

Journ. of Gen. iv 22

336 A Second Brachjidarttjlonx FamUji

The longest middle fingers measure 2 inches (Nos. 8 and 28). The
shortest measures 1^ inches (No. 21), and three measure li inches
(adults).

The tallest male i-s 04.i inches in height (No. 28); the tallest
woman is 59 inches (No. 26) ; the shortest woman 52 inches (No. 9).

B. Feet.

The shortness of the feet is "due solely to the abortivf middle
phalanx, and the somewhat stunted growth of the first."

"The so-called first phalanx of the big-toe is considerably shortened";
in most cases it is reduced to a cube, and in some the shortening goes
even further.

" The middle phalanx in the other toes has become ankyloscd to
the terminal one in every adult." This ankylosis " occurs at an earlier
age in the foot than in the hand, though the hand as a whole is more
abnormal."

"There is an absence of the basal epiphysis of the first, jjhalanv
of the big-toe, though it is conspicuous in the first phalanx of each of
the other toes (in children), so that this bone appears to be really
homologous with the second phalanx of the other toes."

Metatarsus. There is very little wrong with the metatarsal bones,
in which respect they differ from the metacarpals. In two cases (PI. XIV,
Nos. 27 and 28) the third and fourth are the shortest of the series.

Thigh and Leg. These are shorter than normal, but the numbers
especially of the normals are toi> few from which to calculate a reliable
average. There is no doubt, howi;ver, that the short

Stature is entirely accounted for by the shortness of the luwi'r limbs.
When the adults are seen seated they do not strike one as being
particularly short people, but on standing up it is at once apparent
that they are considerably below the average height. The spinal column
is therefore approximately of the normal length.

This fact seems fairly clear fi-om the j)hotographs of the t\\ o brothers
(PI. XI): the one on the right (No. 37) is 17^ years old, 59^ inches in
height, and is Braehydactylous ; the other one is 14 years old, 3 inches
taller, and is not Braehydactylous. The difference in the length of the
legs is very obvious, which no doubt accounts for their difi'erence in
stature.

The infant (No. 48) is three-quarters of an inch taller than his
normal twin sister, in this respect presenting a striking and unexpected

H. Drinkwater 337

contrast to the rule that holds good in adults and youths, so that the
arrest of growth occurs in some later period of life, probably beginning
at or soon after the age of 2 years.

The shortness of hands is well shown in PL XII, Nos. 23, 37 and
41, where they are represented with normal relatives for the sake of
comparison.

Symmetry.

" In every instance the hands are exactly symmetrical, as shown both
by photography and radiograjjhy (PI. XV); and I believe the same rule
holds true with regard to the feet, though both feet were not examined
in all cases." This remarkable symmetry obtains even in those cases
that show special peculiarities of any of the bones, either in shape or
ossification, or in the presence and time of union of the epiphysis.

Persistence.

There is no apparent tendency to revert to the normal type, for
the hands and feet of No. 9 are no more abnormal than those of her
grandchildren ; neither is there any other indication of the abnormality
disappearing : on the contrary the numbers seem to be on the increase,
the abnormals in the last four of the six generations in the English
branch being as follows :

3 in the third generation.

8 „ fourth „

12 „ fifth
23 „ sixth „

One would expect a priori that individuals whose fingers are short,
stumpy and below the average in strength would be so much handi-
capped in the " struggle for existence " that they would be swamped
by the general population, and in an uncivilized state of society this
surely must have occurred ; but the conditions of modern society have
afforded them such chances of survival that their numbers are steadily
increasing, yet it seems clear that they are handicapped to some extent,
for all the employed men and women are engaged in occupations where
there is no great need for manual dexterity : their social position is
below that of their normal relatives.

22—2

;338 A Second Brachijdaclylous Family

Marriiir/e.

Mai-riagu is the thshioii aiiioiigsl. thusu sliorL-fingerod peu])lc :uh\
ihcy " go off," as oin' '<[' the women expressed it, before their normal
relatives. The great iiiajority of those shown in the chart as not
having had children died in infancy. Several adult noi'nials remained
uiimari-ied. Why should this be ? There must be some special fascina-

Measurenwitta of AhimnuaU hi iiichus.

Hand

Initials

in Chart

.Se.v

Age

linger

Lengtli

Wi.ltll

l!lna

Tibia

Heiglil

Mrs E. T. ...

VI '

funuile

64

14

5

3

7i

10

52

Mrs M.

16

,,

37

Is

H

3J

—

—

585

M. H.

17*

,,

33

1*

5

31

—

—

58A

M. H.

18'

,,

31

1^

H

H

9

114

60i

MrsM.A.L...

19'

,,

43

n

H

H

7;

—

55i

Mrs R. F. ..

20*

,,

41

14

19

■i\

9

13

59A

Mrs S. E. J...,

•il*

)i

39

li

4

n

H

9

56

Mrs O.K. ..

23*

,,

26

^

4|

u

74

10^

57J

W. H.

24

male

39

is

—

—

—

—

—

MrsO. G.F...

25*

female

37

14

44

3J

8

"i

57

MrsE. T. ..

26*

,,

28

13

5

U

75

—

59

H. H.

27*

19

!■;

5

31

84

11.^

59

S. H.

28

male

18

2

6

4

64 .^

E. M.

2!)*

female

12S

2

5 J

3

—

—

58

E. M.

HO

,,

lOJ

lis

■414

2;

—

—

52^

J. M.

31*

male

8A

n.

n

n

—

—

474

P. M.

32'

5A

li

H

2|

—

—

42

W.L.

33

„

17

^

54

31

94

—

60.^

E. L.

3.5

M

10| ),

14

44

24

—

—

43

L. L.

36*

female

7

n

4

24

H

8A

43

C. F.

37*

male

l"i

1;

54

H

9

13

594

W. F.

38*

8

^i\.

4

24

63

9

454

E. F.

39*

female

6

1.\t

'^X

2-'

6

7J

424

A. F.

40*

,.

2A

\,

n

2

44

6

32i

S. J.

41*

.,

15

14

5

3

74

11

54.1,

A.J.

42*

:^S

IJ

3

2,V,

5

iij;

32

E. R.

43*

,,

■li

n

■ih

n

5^

8

38J

W.H.

44

male

8

14

—

—

—

—

—

D. H.

4.5

, J

1 1

1

—

—

—

—

. —

J. A. F.

46

12

ij

4i

2S

7.',

11

5U

A. F.

47'

,,

•iV

1,'.

3-:;

2]

«i

8

41

A. F.

48

>>

1>V

"<

n

1:^

34

4i

28J

C. C. T.

49*

,,

7

15

4

24

5S

n

44i

* Individuals marked bj au asterisk are illustrated on Plates XII — XV.

H. Drinkwater

:339

tion that compensates for the manual defects, but what it is I am
unable to say. It might be suggested that their amiability and obliging
disposition (of which this paper is sufficient evidence) will afford an
explanation, but I should not like to say that the normal members fall
short of them in this respect.

Of the first family there are 27 or 28 abnormals now living: so that
altogether there are at least 60 people of the Brachydactylous type in
England at the present time.

The normals do not appreciably differ in the above dimensions from
the general population, and I give the details of only a few children
and adolescents for comparison with their short-fingered relatives.

Measurementg of Normal Members of the Famih/.

Initials

Sex

Age

finger

Lengtli

Width

TTlna

Tibia

Heiglit

A.J.

male

17

H

74

34

94

m

674

W. J.

12

24

6

2|

U

10|

51

A. F.

female

16

2i

6

2|

9

12

58|

F. W.

F. .

male

14

2S

6

3

8i

Hi

54J

L. 0.

F. ..

female

lA

14

3

li

3S

54

274

E. F.

male

19

2J

62-

3*

9

12

6O4

J. P.

,,

14

3

64

34

94

m

62i

K. F.

female

10

24

5|

2|

74

11

50

E. H.

) J

20

3i

7

n

—

—

6I4

H. H.

,,

17

3

7

34

—

—

594

S. M.

male

2i

1|

—

—

—

—

33i

Part of the exj)ense of this investigation has been defrayed by the
" Earl of Moray Fund for Original Research " of the University of
Edinburgh. I veiy gratefully record my thanks to the Trustees for
their generous help.

340 A Second Brarfn/dactylous Family

EXPLANATION OF PLATES.

The numbers are those given in the chart on p. 3'J7 and also in the Tahle on p. 338.

PLATE XI.

i'ig. 1. Shows three braclijdactylous females ; a cliild, its motlier and grandmother
(No. 43, 23 and 9). The sliort stature of the women is evident when compared with
that of the author which is 5 feet 8?. inches.

Fig. 2. Shows two brothers. The one on the right is brachydactylous and two years
older than tlie one on tlie left, who is normal.

PLATE XM.

Photographs of hands and a foot. The numbers refer to those in the cliart. Their ages
and other particulars are given in the table of measurements on page 338. The same
applies to the following plates.

PLATE XIM.
All these hands are brachydactylous.

PLATE XIV.
Eadiographs of hands and feet, all abnormal.

PLATE XV.

Radiographs of hands. Both hands are shown in order to illustrate the bilateral
symmetry.

JOURNAL OF GENETICS, VOL. IV. NO. 4

PLATE XI

JOURNAL OF GENETICS, VOL. IV. NO. 4

PLATE XII

.No. 2:1 and normal.

No. S7 and mirmal.

Nu. -JU.

No. 43.

No. 311.

Xo. 27.

No. 41 and nouual.

JOURNAL OF GENETICS, VOL. IV. NO. 4

Nd. 'J.

^o. -21.

No. 17.

No. ■>:i

No. is.

No. 2.5.

No. ly.

.No. -Jlj.

PLATE XII

N"o.

No. i-2.

No. 30.

No. 43,

No. 38.

No. 47.

No. 4U.

No. 4'J.

JOURNAL OF GENETICS, VOL. IV, NO. 4

No. 20.

Jfe^^P^^

Nu. S9.

Xo. 37.

PLATE XIV

No. -21).

No. 27

Xo. 31.

No. 41.

JOURNAL OF GENETICS, VOL. IV. NO. 4

No. 1«.

Ku. 27.

PLATE XV

No. 29.

v*S.

No. 31.

ON THE PRIMARY AND SECONDARY SEX CHAR-
ACTERS OF SOME ABNORMAL BEGONIA
FLOWERS AND ON THE EVOLUTION OF
THE MONOECIOUS CONDITION IN PLANTS.

By C. J. BOND.

PART I.

My attention was first directed to the problem of Sex Dimorphism
in the Begonias when noticing that certain flowers in which the process
of Sex Differentiation was incomplete were almost invariabl}^ accom-
panied by an abnormal or supernnraerary Floral Bract. This accessoiy
and asymmetrical Floral Bract occurs on the pedicle which bears the
abnormal flower. It seems to mark the situation in the growth of the
flower stalk where under normal conditions the Male and Female Sex
Organs are segregated out into flowers of opposite sex. (PI. XVI, fig. 1.)

The importance of this abnormal floral bract lies in the indication it
affords of the fact that the differentiation of the Sex Character is a
matter of qualitative cell division in somatic as well as in germinal
cells.

The next point is that partial or complete failure to undergo quali-
tative cell division in these abnormal flowers is associated with abnor-
mality of another kind. This secondary abnormality may take the form
of modification in, or increased number of accessory floral parts, or
elongation of the staminal axis with increased number of stamens, or
petallody of stamens (doubleness in the male flower). In the female
flower it may take the form of an increased number of carpellary leaves,
as indicated by an abnormal number of ovarian loculii, or (by failure of
these carpellary leaves to close) an exposed condition of the ovules, a
partial return in fact to the Gymnospermous condition.

Any of these changes may afford the earliest indication in male and
female flowers respectively of a distuibance of sex equilibrium, in other

342 Sex Characters in Begonias

words a disturbance in the regularity of the qualitative cell divisions by
which the male and female sex organs are normally differentiated out
into different flovv'ers.

A further stage in abnormality may be the appearance of male
gamete bearing organs on female flowers, or more rarely the appearance
of female gamete bearing organs on male flowers.

Finally, a complete ring of stamens, functionally active, may surround
the gynoecium of a flower of female type. (PL XVI, fig. 7.)

This association in the same flower between abnormalities of ac-
cessory and of essential floral parts is important because it shows that
instability of equilibrium in the primary sex elements carries with it in
nearly all cases instability of equilibrium of somatic tissues also.

The association between sexual instability or hermaphroditism and
modification of accessory floral parts is also shown genetically thus: —
The Fi seedlings of a cross between a Begonia with double male flowers
as the seed parent and a single flowered variety as the pollen parent
are for the most part single flowered plants. A certain number, how-
ever, show indications of a tendency to doubleness by the presence of
one or two extra petals in the male flowers, while others exhibit an
elongation of the axis which bears the otherwise normal stamens. In
other words singleness is only partially dominant over doubleness in the
F^ generation of such a cross.

Of the seedlings of the F., generation so raised an abnoimal per-
centage shows a tendency to hermaphroditism and abnormal flowers.

It is more difficult by cross fertilization to transfer the ddiible
character to female flowers, that is to convert a sex limited character
like doubleness in the Begonias, into a specific character. In the female
as in the male flower however the earliest indication of sexual instability
or hermaphroditism is often the presence of an increased number of
pistils and carpellary leaves. Thus in plants (as in animals) any disturb-
ance in the structural if not in the functional characters of the primary
sex organs is usually associated with changes in the secondary flur.il
characters.

Primarily Male and Primarily Female Flowers.

The question now arises whether the arrangement and distribution
of the male and female sex organs in these hermaphrodite Begonia
flowers throw any light upon the central position of the Ciyno(>cinm in
our modern hermaphrodite flowering plants.

C. J. Bond 343

It is a matter of considerable interest to find that two types of
abnormal flowers exist in the Begonias : —

1. The common type in which, round a central more or less per-
fectly developed gynoecium, rudimentary organs of the male type occur.
(PI. XVI, fig. 7.)

2. A far less common type in which rudimentary female organs are
gathered round a centrally placed androecium. (PI. XVI, figs. 3, 4, 5, 8.)

Flowers of the first type are here called primarily female. Flowers
of the second type are called primarily male. The criterion being the
relative position of the male and female sex organs to each other on the
common floral axis.

It is here suggested that the almost universal central and terminal
position of the female organs in hermaphrodite flowers is a fact of
considerable phylogenetic importance.

It would seem that the female portion of the flower represents the
undifferentiated reproductive rudiment from which the male portion
has during evolution segregated off.

This suggestion receives some support from the frequent (though
not invariable) occurrence of vestigial remains, or rudimentary organs
of the opposite sex, in the female rather than in the male flower in
monoecious and in some dioecious plants. It would be interesting to
know how many of these cases in which vestiges of the opposite sex
organs are found in the male and not in the female flower represent a
return to, rather than a step forward towards, the hermaphrodite form.
Further the question of the homozygous or heterozygous nature of the
male and the female plant, in respect of sex in the Mendelian sense,
assumes additional importance from this point of view.

Bearing in mind the fact that sex difterentiation is one of the
earliest stages in plant, as in animal evolution, and that the difteren-
tiation of male and female sex organs on different individual plants or
on different parts of the same individual plant, was probably already
established in the Cycads, Ginkgos, Sphenophylls and other possible
ancestors of our flowering plants, the problem of the sex evolution of the
hermaphrodite flower is probably not so much a question of differentiation
of sex organs into male and female, as of bringing together into close
juxtaposition on one common floral axis, male and female sex organs
formally located in different plants or in different parts of the same
plant.

Though the active factor in bringing about the evolution of the
hermaphrodite flower was probably a necessity for adaptive capacity on

.144 Sex Characters in Beffonias

the p;ut 111 phiiils Lo iindcrgu fertilization by means of insect instead of
wind agency, in association with the great evohition of insect life which
occurred in cretaceous and precretaceous times, the essential factor
which determined this adaptive change along primarily' female rather
than primarily male lines was probably, not any superior capacity for
fertilization so obtained (though this may have been a point in its favour),
but the greater facility afforded by the central position of the gynoecium
on the floral axis in providing for the longer retention of the embryo
in contact with the tissues of the parent plant.

This growing tendency to retain the embryo in contact with the
mother plant, and thus provide it with a better start in the world, forms
a striking feature of plant development in recent times, just as the
tendency to retain the embryo in contact with the maternal tissues is a
marked characteristic of the mammalia, the highest order of animals.

The ultimate aim, if one may be allowed the teleological expression,
has been the production of the largest number of offspring with the best
chances of successful separate existence, along with the greatest economy
of material, and the chief steps towards this end have been: — (1) The
replacing of the naked ovule by the ovule enclosed in an ovary, i.e. the
evolution of the Angiosperms. (2) The provision of nourishment in an
available form to as late a stage as possible in the growth of the embryo,
and this was secured by placing the ovary in the centre of the floral
axis.

The provision for the passing of the jirothallus or asexual repro-
ductive stage while the embryo remains in contact with the maternal
tissues is also in harmony with this scheme.

The ultimate test of the respective value of the unisexual as against
the hermaphrodite type of flower, is capacity to ensure, not merely the
fertilization of a large number of ovules but the survival of a large
number of embryos, and the central position of the gynoecium in the
hermaphrodite flower favours such survival.

In this connection one is reminded of Baur's conception of hybrids
as Clinal and Periclinal Chimaeras. It is possible to apply this con-
ception to the hermaphrodite flower which would then be a sex-chimaera
built up on a clinal or central female basis with periclinal male accessory
organs.

The problem of the central position of the gynoecium in the her-
maphrodite flower when I'educed to cytological terms becomes a question

1 "Primarily" iu the sense described above in reference to the position of tlie
gynoecium.

C. J. Bond 345

of the relative position assumed by the factors for maleness and feinale-
ness in those qualitative cell divisions which control the segregation of
the sex organs. Thus if maleness passes into the lateral daughter cells,
while femaleness is retained in the cells which continue the axial line
of growth the flower although hermaphrodite is primarily female. If
on the other hand (as in these abnormal Begonia flowers) femaleness
is thrown off into the lateral daughter cells and maleness remains in
the central cells then the flower although still hermaphrodite is primarily
male.

It is in fact even more true of plants than animals that sex is a
function of the position of the factor or factors which control it.

These abnormal Begonia flowers, primarily male and primarily
female, also show that under certain conditions the qualitative cell
divisions which determine sex may be imperfect or incomplete. Thus
if the type of the division has been settled by the passage of the factor
for maleness into the central daughter cells, then any remnant of the
female factor if present must apparently pass into the lateral daughter
cells. This fact suggests some association between the volume of the
ftxctor and the position assumed by that factor in cell division. The
conception is indeed forced upon us that the sex of a unisexual flower
on a monoecious plant partly depends on the relative volume of the sex
factors present in that flower. Whether the male organs shall occupy
a central or a circumferential position on the floral axis (in other words
whether the flower shall be male or female) seems to depend partly on
whether the factor for maleness is present in greater volume than the
factor for femaleness. If it is, then maleness is handed on to the
daughter cells in the direct line of growth and the flower is primarily
male, whereas if the volume of the female factor is greater than that of
the male factor, then femaleness continues in the line of growth and
maleness is cast off into lateral daughter cells.

The importance of this matter lies in the fact that sex segregation
in the flowers of monoecious plants seems to be associated with a definite
position assumed by the alternative fixctors which control sex either in
one or in a series of qualitative cell divisions.

One may perhaps hazard the suggestion that if future cytological
research should associate the male (or the female sex) in the Begonias
with the presence of an accessory chromosome, then the position
taken up by that accessory chromosome in the qualitative cell divisions
which divide the growing cell into end on and lateral daughter cells
will determine the primary sex of the flower, just as the passage of such

34G Sex Characters in Begoniaa

an accessoiy chromosome into one of two alternative gt'iiii cells deter-
mines the sex of the individual. At present we are ignorant as to the
stage in the growth of the floral parts at which this segregating division
occurs. Neither do we know the difference in nuclear or factorial con-
.stitution between the primarily male and the primarily female flower oi-
whether this difference is of a genetic or a somatic character.

Primanly Male and Primarily Female In florescences.

This concef)tion of a flower as primarily male oi- primarily female
may also be extended to the Inflorescence.

In the normal inflorescence of the tuberous Begonia the flower
which terminates the floral peduncle is a male flower. A few abnormal
examples occur in which on cui-sory examination it appears that a
female flower terminates the inflorescence, but careful inspection will
show that even in these cases a rudimentary or abortive male terminal
flower really exists. If in some varieties with double male flowei-s the
inflorescence is apparently entirely composed of one male flower, careful
examination will reveal however rudimentary or abortive female flowers
in the axils of the bracts of the floral peduncle which carries this flower.
Further, in some varieties both in the tuberous and fibrous kinds
(e.g. Mrs J. Heal, Beg. Socotrana, Gloire de Lorraine, and others) only
male flowers are present. In these cases the inflorescence is not
symmetrical and tripartite, but asymmetrical with male flowers given
oflf on one side only. In some climbing Begonias the male flowers
nonnally appear at the fourth or fifth dichotomous division of the floral
peduncle while female flowers appear later at the sixth or seventh
division; further, as the male flowers fall early the result is that the
inflorescence appears to be composed of female flowers only. The point
of physiological and cytological interest about the inflorescence thus
becomes (as in the flower) a problem of the relative position of certain
organs. In other words the disposition of sex organs in flowers of
opposite sex like the disposition of sex organs on a ccmimon floral
axis in the hermaj^hrodite flower is a problem of qualitative cell division.
In the inflorescence, as in the flower, we find two types : —

(a.) A male type in which the terminal flower is a male flower and
the female flowers are thrown oft' laterally, as in the Begonia.

(6) A female type in which the terminal flower is a female flower
and the male or hermaphrodite flowers are thrown off laterally, as in
some caryophyjlaceous plants.

C. J. Bond 347

The type of inflorescence and the type of flower may vary in the
same individual plant, and the question arises whether the Begonia
(and other monoecious plants) may not have attained their present
monoecious condition in two ways: (1) by diverging from the herma-
phrodite stage along primarily female lines as far as the flower is concerned,
and (2) by developing along primarily male lines as far as the inflor-
escence is concerned. In the walnut and some other monoecious trees
the occasional occurrence in some individual trees of a rudimentary
gynoecium in a central position in a male floret, suggests that these
plants have passed through a hermaphrodite stage in which the flowers
were built up on a primarily female type.

Sex Dimorphism thus becomes a problem of (1) the kind of units
among which the sex differentiating process takes place, and (2) the
period in the life history of these units at which this process occurs. If
the diffei-entiating cell division occurs in the germ cell stage the
dioecious variety of sex dimorphism results, if it occurs a little later
during the development of the flowers the monoecious form results, while
if it occurs at a still later stage during the development of the sex
organs on the common floral axis the hermaphrodite flower is formed.
We are ignorant of the factors which determine the stage at which this
all-important segregation shall occur, but of the three stages at which
it has up to the present taken place in the history of plant evolution,
the middle or monoecious stage seems to have been the most unstable
of the three. For it is in monoecious plants that we find the greatest
number of vestigial remains or rudimentary organs of the opposite sex.
In some monoecious plants, e.g. the Box, indications exist that the
plant is now on the way to or away fi-om the hermaphrodite condition.

It is a matter of considerable interest to find that in plants as
in animals a close association exists between a sex abnormality like
hermaphroditism and a delayed occurrence of those differentiating cell
divisions which determine sex. Postponement of the sex differentiating
cell division from the germ cell to the zygote stage may not only bring
about hermaphroditism in the individual, but the actual period in the
development of the zygote at which the differentiation of sex organs
occurs also influences the type of hermaphroditism which results, for
instance, if the differentiation of the factors for maleness and femaleness
is postponed to the stage of blastomeric division, in which the right and
left halves of the animal body are laid down, then a lateral gynandro-
morph is produced. Many such cases have been recorded among insects,
and quite a number have now been recorded among the vertebrates.

348 JSex Characters in Begonias

especially in birds. (See H. Poll, "Zur Lehre von den sekundaren
Characteien " (Stzgtibei: Ges. Naturf. Freunde zu Berlin, 1909) ;
C. J. Bond, "On a case of Unilateral Development of Secondaiy Sex
Characters in a Pheasant" {Journal of Genetics, Feb. 1914).) In this
connection also the limitation of paternal and maternal characters to
opposite sides of the body in the larvae of some sea urchin hybrids
reared by Herbst as the result of artificial fertilization is of great
interest. (See J. Loeb, Artificial Favthenogenesis.) Although I am
not aware of any recorded observations of the limitation of paternal and
maternal characters to apposite halves of the individual in the cotyle-
donary .stage in seedling plants, yet such limitation does undoubtedly
occur at a later stage of growth, for both individual leaves and opposite
branches may sometimes show a hemilateral arrangement of paternal
and maternal characters.

Certain conditions in animals suggest that in addition to the
bilateral type of architecture, the individual organism is also built up
on a serial or segmental plan, thus sex abnormalities like hermaphro-
ditism may show a segmental distribution in birds. (C. J. Bond,
Journal of Genetics, Feb. 1914.) In plants also the distribution of the
unisexual florets in the compound inflorescence follows in some abnormal
cases a distinctly segmental or periodic type. This is well seen in the
irregular inflorescence of some abnormal maize plants. Plate XVII,
fig. 1, shows a serial alternation on the same peduncle of male and
female florets. Plate XVII, fig. 2, shows a magnified view of the same

th

ng.

PART II.

Secondary Sex Characters in Begonias.

It has already been pointed out that the normal male flower of the
monoecious Begonia has four petals and the normal female flower five.
Although this association between the sex of the flower and the number
of petals seems characteristic of most Begonias under normal conditions,
yet it is not an absolutely constant feature. For instance a flower may
develop an outer whorl of pistillate structures on its staminate floral
axis. It may even show exposed ovules on some of these pistillate
leaves, it majf in fact be partly a female flower and yet retain the four
petals characteristic of the male flower. (See PI. XVI, figs. 8, 4, 8.) In
the same way the jjrimarily female flowei' may have a row of .stamens

C. J. Bond 349

outside the central pistil and yet retain the five petals characteristic of the
female flower. (PI. XVI, fig. 6.) Although doubleness chiefly affects male
Begonia flowers, and is therefore a sex limited character, it is possible
under certain conditions, by cross pollination, to transfer this character
of doubleness to the female flower. Double female flowers so produced,
however, retain secondary male characters. The structure and arrange-
ment of the accessory floral parts are not influenced by being linked up
with maleness. In fact a primarily female flower with a well-formed ovary
and a central gynoecium may develop a complete ring of stamens and yet
retain the five petals characteristic of the female flower. (PI. XVI, fig. 6.)
From these facts we are I think justified in concluding that the presence
of male organs does not necessarily modify the secondary sex characters
of female flowers. The same is true of male flowers which develop
female organs. The secondary characters seem to depend on the
primary sex of the flower as shown by the central position of the
gynoecium or androecium on the floral axis. The accidental occurrence,
so to speak, of rudimentary, or even well developed, organs of the
opposite sex, does not affect the growth of the secondary sex characters.
That this should be so is not surprising when we recall the apparent
absence in plants of any secretion corresponding to the sex hormones
in animals.

There is, however, evidence pointing to the existence of enzymes in
plants.

Keeble (Plant Animals, 1910) suggests that the cambium cells
influence the neighbouring cortex cells and stimulate them to undergo
cell division by means of some secretory substance. He also suggests
that the green algal cells in Convoluta roscoffensis supjjly the bio-
chemical stimulus on which the later development of that organism
depends, and in the absence of which it fails to grow.

There are also the important facts concerning the influence of fer-
tilization on the growth of accessory floral structures. Darwin showed
long ago that some kinds of foreign pollen can act as a poison to the
stigmata and pistils of certain flowers. The rapid wilting and the
falling within 36 hours of the petals of a successfully fertilized female
flower provides us with a practical indication as to whether the pollen
artificially introduced has " taken," and whether the flower is adequately/
fertilized in any given case. But these latter examples are examples
of an inhibitory rather than a stimulatory influence. In the other
direction we have the stimulating eflect of the fertilized and growing
ovule on the tissues of the floral peduncle, and this influence is

350 Sex Characters in Begonias

transmitted through the tissues which connect the ovule with the
mother plant.

In the dioecious Lychnis if pollination and fertilization of the ovules
be prevented the female flower falls on the fifth or sixth day after
opening. This falling of the unfertilized flower (like the falling of the
autumn leaf) is brought about by a degenerative change in the collar
cells which form the neck of the floral peduncle at the point where this
swells out into the thalamus or cone on which the ovules are imbedded.
If on the other hand the ovules have been fertilized active growth
occurs in these cells. Either the absence of a stimulus derived from
the growing ovules or possibly the presence of an inhibitory sub.stance
formed by the atrophying and degenerating ovules brings about the
death and degeneration of these collar cells, and this results in the fall
of the unfertilized or insufficiently fertilized Hower.

In the falling corolla after pollination, and the falling flower in the
absence of pollination, a quantitative relationship can be observed
between the number of pollen grains which gain access to the pistil
and the rapidity of the wilting of the stigma and corolla in the one
case, and the number of ovules which undergo fertilization and the
rapidity of the falling of the flower in the other. If only a few pollen
gi-ains are allowed to gain access to the pistil the petals may remain
erect and vigorous for several days, while if the stigmata be dusted with
a larger quantity of pollen they fall in 24 to 48 hours. In the case of
the flower, if only 5 or 6 ovules (in Lychnis) undergo fertilization the
degenerative change in the collar cells may not be prevented and the
flower may fall although it has been partly fertilized. A minimaJ
number of ovules must in fact undergo fertilization and begin to grow
in order to prevent those changes in the cells at the root of the thalamus
which lead to the falling of the unfertilized flower.

This fjict, viz. that a quantitative relationship e.xists between the
factor which stimulates or arrests cell growth and the stimulated or
arrested cell activity which results from its influence is an important
one. It is unlike the behaviour of an animal enzyme which when
introduced into a fermentable solution serves to hydrolyse its whole
volume.

It is also unlike the method of action of speciflc sex hormones,
where the .smallest quantity of the substance, if present in the blood
stream, is capable of influencing the metabolism of the whole body.

In fact these chemical substances which stimulate and arrest cell
growth in plants seem to be incapable of self multiplication and seem
to exercise only a local influence on the tissues of the plant.

C. J. Bond 351

PART III.

The question now arises whether the factor, or factors, which
control sex segregation in the monoecious plant are the same as those
which determine sex in dioecious plants and in animals.

In animals and dioecious plants segregation of the factors con-
trolling sex takes place among germ cells either before or during
fertilization, whereas in the Mowers of monoecious plants segregation
takes place among cells which present the external characters of somatic
cells although they no doubt retain germinal material capable not only
of forming new individual plants by bud formation, but also of acting
as the carriers of hereditary characters.

But both processes depend essentially on qualitative cell division.
In one case this takes place in the meristematic cells of the plant stem
and results in maleness passing into the terminal and femaleness into
the lateral daughter cells, or vice versa ; in the other case it occurs
amongst male and female bearing gametes at an earlier stage of their
life history, probably at the period of maturation.

The chief distinction between the two processes is one of time of
occurrence rather than one of kind. One affects the germ cell at or
before fertilization, the other affects the zygote which results from the
repeated division of this germ cell after fertilization.

It seems clear that the association between the segregation of
primary and the segregation of secondary sex characters is less intimate
in plants than in animals. Both depend on qualitative cell division,
but the primary sex organs do not exercise such continuous control
over and are not so indispensable to the growth of the corresponding
secondary sex characters in plants, as in animals. In the higher
animals, unlike some invertebrates, a very elaborate system of inter-
dependence and control is present between those cell divisions which
represent primary maleness and femaleness and those metabolic pro-
cesses which underly the production of male and female secondary sex
characters. The functional activity of the latter is dependent on the
functional activity of the former, whereas in the case of the monoecious
plant the determination of the sex of the flower, and the number and
arrangement of its petals, although both may be primarily under the
control of a common genetic factor, are more or less independent
processes.

Journ. of Gen. iv 23

352 Sex Characters in Begonias

EXPLANATION OF PLATES.

PLATE XVI.

Fig. 1. Shows to the liglit an abnormal hermaphrodite flower in association with an

asymmetrical floral bract.
Fig. 2. Shows "exposed ovules'" in a partially hermaphrodite flower.
Fig. 3. Shows female sex organs, ovules, in a peripheral position on a primarily male

flower. (See also Fig. 8.)
Fig. 4. A primarily male abnormal flower with a ring of pistillate structures peripheral to

the central androecium. This flower, although partly hermaphrodite, preserves the

four petals characteristic of the male flower.
Fig. 5. A ijrimarily male flower partly hermaphrodite with oue or two pistillate structures

periijherally situated on the floral axis. This flower had four petals only.
Fig. 6. Abnormal primarily female flower with a peripheral ring of stamens round a

central gynoecium and the Ave petals characteristic of the female flower.
Fig. 7. Two primarily female hermaphrodite flowers. A peripheral ring of stamens

surrounds the gynoecium in each case.
Fig. 8. Terminal primarily male flower with abnormal stamens, multilocular anthers,

and ovules peripherally placed on the petals. Though partly hermaphrodite it has

the four petals characteristic of the male flower.

PLATE XVII.

Fig. 1. Inflorescence of maize plant showing a serial alternation on the same peduncle
of male and female florets.

Fig. 2. Magnified view of same.

JOURNAL OF GENETICS, VOL. IV. NO. 4

l-V- 1.

Fig. -2.

i'lg. -i.

PLATE XVI

Fii'. i.

msm

'4

^

Fig. 5.

Fig. 0.

JOURNAL OF GENETICS, VOL. IV. NO. 4

PLATE XVII

ON THE OKIGIN AND BEHAVIOUR OF
OENOTHERA RUBRICALYX.

By R. RUGGLES GATES, Ph.D., F.L.S.,

University of London.

In a recent paper which purports to deal with F^ hybrids of
Oe. rubricalyx, Shull (1914) takes occasion to attack my conclusions
regarding the origin and hereditary behaviour of this mutant. I may
remark that I have been studying the hereditary behaviour of Oe. mut.
rubricalyx ever since it appeared in my cultures in 1907. The results
of these studies, portions of which have been published in several papers
(1909 — 1914), are perfectly clear and definite, and the main conclusions
to which they have led are, in my opinion, irrefutable. Among my
crossing experiments is an extensive series of ^i and F^ hybrids
be4;ween ruhricalyjc and Oe. grandiflora, Solander (Gates, 1914) num-
bering nearly 3000 plants. I have also gi'own a still more extensive
series of F-^ families, whose results confirm the previous conclusions
from the Fo, families and will be summarized in a book which is now
in press.

Before entering further into the evidence on which my conclusions
rest, it may be well to examine for a moment the nature of the evidence
which Shull brings forward as the basis for his criticism and for con-
clusions which are contrary to mine. In the first place, the internal
evidence from the author's own paper, as well as other facts, shows that
his supposed pure rubricalyx was in reality Oe. rubricalyx x grandiflora.
This in itself of course vitiates in toto the " results " stated in his paper,
for in every cross mentioned one must read " rubricalyx x grandiflora"
instead of "rubricalyx." For example, when he describes what he states
is rubricalyx x rubrinervis F^ he is really describing (rubricalyx x g7-a7i-
diflora) x rubrinervis. What must one think of observations which
pretend to be critical, and yet which make the fundamental error of

23—2

354 Oritjin and Behaviour of Oenothera rubriealyx

failing to distinguish between pure rnhncalyx and its iiybrid with a
wholly distinct species ? These two things are so different that the
veriest amateur could have no difficulty in distinguishing between them,
either as rosettes or as mature plants.

Such looseness of observation as ShuH's paper displays is inexcusable
when put forward with such a show of confidence in the soundness of
his results. I have stated repeatedly in previous papers that rubncalj:';
is morphologically identical with rubrinervis, of which it is in fact a
very marked and striking colour variety. Photogi-aphs have also been
published (see Gates 1911, Figs. 4 and 5) which prove these assertions.
However, Shull evidently had occasional misgivings regarding the real
nature of his supposed rubriealyx rosettes, as when he says (footnote,
p. 84) : " Several features of my plants suggested a relationship to
0. grandijtora, particularly the rather lax rosettes, strong red spotting
of the young rosette-leaves, and the development of buds more slender
and rounder than in my 0. rubrinervis cultures." As I pointed out
several years ago (1909), these are all conspicuous features of Oe.
grandiflora, and this knowledge should have given him pause, par-
ticularly when he was aware that his " ruhriwlyx " plants were fi'om
unguarded seeds.

In several places through his paper Shull wavers between the
assumption (which he knew was doubtful) that his " rubriealyx " was
pure, and the admission that it may have been a cross with yrandifiora.
Yet he must have realized that his results were almost valueless if the
latter was the case, and quite so if he was unable to determine between
these two alternatives. It is difficult to understand how the paper
coidd have been brought forward as a contribution to genetics under
these circumstances. Since the author states that it was originally
written before my last paper (Gates, 1914) dealing with rubriealyx
and its hybrids was published, the references to that paper, as well
as certain other changes, must have been made later.

The plants which Shull assumed to be pui'e rubriealyx were obtained
by him from Dr A. F. Blakeslee, of Storrs, Connecticut, who grew them
from unguarded seeds of rubriealyx received from me. Dr Blakeslee
has kindly sent me the original seed packet, from which I find that the
seeds in question were collected from a plant grown in 1909. I grew
several families of rubriealyx in that year, and one of these was adjacent
to a culture of grandijloi-a. These unguarded seeds were not intended
for genetic experiments, and were sent (marked " open-pollinated ") in
answer to a request for any plants which would show the red-budded

E. RuGGLEs Gates 355

character for demonstration purposes. In using them for breeding
experiments, knowing they were unguarded, Shull took his own chances,
with unfortunate results. Though I did not record on the packet the
pedigree number of the plant from which these seeds came, I have
other reasons than the subsequent results for believing that they were
from the culture next to the grandiflora plants, where the opportunities
for crossing with that species were of course greater. So far as can be
learned, Shull's plants were probably derived from seeds directly from
this packet, in which case they would be r uhri calyx x grandiflora, F^.
I have already (1914) published an extended account of several years'
experiments with this cross and the reciprocal in F^ and F.,, as well
as sesquireciprocal and double reciprocal crosses. Although the same
strain of Oe. grandiflora was not available for all these crosses, yet
all the features of buds, foliage and development were the same in
the F., families from ruhricalyx x grandiflora and grandiflora x ruhri-
calyx, so that any disadvantages from this source are more theoretical
than real. A glance at Shull's published figures of his "ruhricalyx"
shows clearly by their bright red spots that they have been crossed
with grandiflora, and the leaf-shape also shows some effect of this cross,
even in the young rosettes.

Since the differences between Shull's conclusions and mine result
from the fact that I was dealing with ruhricalyx while he experimented
with ruhricalyx x grandiflora under the misapprehension that it was
ruhncalyx, his conclusions obviously fall to the ground, and his
criticisms of my experiments on the basis of his "i-esults" are worth
nothing.

Nevertheless, the origin of ruhncalyx is a matter of such signifi-
cance and has been so much discussed that it seems worth while to
restate the evidence on which rests my conclusion that ruhricalyx
originated from ruhri7iervis through a single unit-change. The ulti-
mate nature of this change will be discussed in my forthcoming book.

In his anxiety to find a basis for doubting that ruhricalyx originated
as a simple Mendelian dominant, Shull does not treat the observed data
faii-ly. Instead of considering all the data, he omits entirely certain of
the, ratios which disprove his unsupported assumption that ruhricalyx
can have originated through the union of two independent factors for
red. The data, if all taken into account, are however ample to show
that ruhricalyx when it appeared . did actually differ from ruhrinervis
in a single Mendelian factor. It is, indeed, decidedly amusing to find
a Mendelian repudiating his own method of argument when it happens

356 Oriffin and Behaviour of Oenothera rubricalyx

to lead to conclusions which he docs not wish to accept. But if the
Mendelian argument holds in nthcr cases (as I think it docs), then it
will have to be admitted to hold also in this case. The significance of
mathematical ratios can no more be suspended for the convenience
of some one, than can the law of gi-avitation. I hold no brief for
Mendelism, but when the fiicts point so clearly to a 3 : I ratio in
the offspring of the rubricalyx mutant, and a 1 : 1 in the cross of a
heterozygous rubricalyx individual with another species, I am not loath
to draw the inevitable conclusion that the mutation originated through
a single dominant unit-change antl that heterozygous plants were pro-
ducing two types of germ cells in equal numbers.

The evidence is as follows : The offspring of the original rubricalyx
mutant which were not accidentally destroyed numbered only 12. Of
these, 10 came into bloom, and 9 of them were shown by their buds to be
rubricalyx and 1 ruhrinervis. Two other plants remained losettes, and
I formerly classed them as undetermined or doubtful because they
showed only a small amount of "ventral" red. Subsequent examination
of many rosettes, however, has shown that whenever there is even a
trace of ventral red on the rosettes the plants invariably develop red
buds. Hence all rosettes which were formerly classed as doubtful
because they showed only small amounts of ventral red, were really
rubricalyx. The above ratio in the F^ of the 7-vhricalyx mutant was
therefore 11 rubricalyx : 1 rubrinervis. This number is of course too
small to determine a ratio. But three of the rubricalyx plants in this
culture were selfed and yielded F.^ families containing rubricalyx and
rubrinervis in the ratios respectively of 10 : 5, 14 : (5 and .33 : 11. These
three ratios are all very closely in accord with 3:1, and even the 11:1
ratio is not a wide departure. Adding these four ratios we have a
total ratio of 68 : 28, which is extremely close to a 3 : 1 ratio, while the
chances against it representing a 15 : 1 are enormous.

This evidence in itself is clearer and more indubitable than that
on which many conclusions from Mendelian ratios rest. But there is
still more evidence, which Shull omits to quote. Two of the rubricalyx
plants in the F^ culture which gave the ratio 33 : 11 and thereby
proved the monohybrid character of rubricalyx, were crossed reci-
procally with Oe. (jrandiflora. They both proved to be heterozygous
in regard to a single character-difference, for in both cases the F^
contained about 50 % red-budded and 50 % green-budded plants. Thus
in rubricalyx x grandijlora, of 67 plants 58 came into bloom, and of
the latter 30 were red-budded and 28 green-budded. This is a far

R. RugCtLES Gates 357

closer approach to equality than Mendelians frequently regard as
sufficient to prove their point. In the reciprocal cross, yrandiflora
X ricbr{calyx\ the F^ contained 147 plants, of which 58 bloomed. The
latter were in the ratio 34 red-budded to 24^ green-budded. Anyone
can decide for himself whether this ratio is nearer 1 : 1, as it should
be on my view, or 3 : 1 as it should be on Shull's view. The full data
from this femily, however, prove the situation still more conclusively,
for all the plants were carefully examined in the rosette stage and 42
were found to show clearly red on their ventral surfaces, 71 were
without any trace of red, and 37 were doubtful, i.e. showing the red
faintly. We now know that the latter must be classed with the first,
for all alike develop red buds, and plants with very little red on the
rosette have the usual deep red buds. In this family there were there-
fore 79 with the ruhricalyx character {R) to 71 withotit it ()•), a close
approach to equality. •

These ratios are exceptionally close to expectation and, taken
together, they furnish abundant evidence as will, I think, be agreed
by all Mendelians, to establish the very high probability that a single
unit-difference was concei'ned in the origin of ruhricalyx from rubri-
nervis. On the other hand, the evidence is sufficient to establish
absolutely the impossibility that two Mendelian fixctors could have

been concerned. These

facts

are shown in

the

following Table

Actual ratios

Expectation
on autlxor's view

Expectation
on Shull's view

11 : 1

9 : 3

11-25 : 0-75

10: 5

11-25: 3-75

14 06 : 0-94

14 : 6

15 : 5

18-75: 1-25

33 : 11

33 : 11

41-25 : 2-75

Total ... 68 : 23

68-25 : 22-75

85-31 : 5-69

30 :28

29: 29

43-5 : 14-5

79 :71

75 :75

112-5 : 37-5

From this Table it will be seen that there is not an atom of
evidence for the presence of duplicate factors in Oe. ruhricalyx, while
the evidence for a single factor is of the kind which is universally
regarded by Mendelians as conclusive.

In addition to the records presented above, several other ratios
were given in my paper (1914, Pedigi'ee 1). These are of families

' A full account of these crosses is given in my former paper (1914).

^ In a previous paper (Gates, 1914, Pedigree 1) this was given as 23. I have not the
original records at hand for determining the cause of this discrepancy, but the difference
is immaterial to the argument anyway.

358 Origin and Behaviour of Oenothera rubricalyx

descended from rahricnlyx individuals in the cultuu' having a ratio
of 33 : 11. They were all observed only as rosettes, and as they were
not observed with the same care as the family of 147 above-montionod
it is obviously unsafe to attempt to draw any conclusions from them.
It is certain, however, that the number of rubrinervis rosettes was over-
estimated, for the reasons given earlier in this paper. As recorded, the
seven cultures total 91 R : 78?-. In any case the numbers do not support
Shull's hypothesis of duplicate factors.

Turning now to certain other statements in Shull's paper, we find
(p. 8.5) the statement "Gates (1914) wavers between the treatment
of the ruhricahjx type of pigmentation as a Mendelian and as a non-
Mendelian character. His most positive declarations on the subject
are that it is non-Mendelian ; but if he sincerely {sic) holds to this
conviction it is strange that he should continue to treat the genetic
behavior of this character as if it 'threw valuable sidelights on Men-
delian phenomena." It is difficult to realize the frame of mind in
which such a statement could be written, showing as it does entire
failure to grasp the points of view developed by me in the paper in
(juestion. One can only conclude, as other statements also indicate,
that it was not carefully read or digested.

Among the "facts" presented in Shull's paper we find the statement
that the offspring of his " rubricalyx " (which we now know to be
rubricalyx x grandifloru) yielded (in F., or a later generation) 117
individuals, of which 107 were red-budded and 10 green-budded.
This ratio, 10'7 : 1, is rather close to the ratio 9'.5 : 1 obtained by me
(1914, p. 235) in an F., family of grandifiora x rubncalyx numbering
157 plants, and confirms my results in obtaining various aberrant ratios
from these ci'osses. It is astounding to find that although Shull is
unwilling to recognize 33 : 11 as a 3 : 1 ratio, yet he is capable of sug-
gesting that 107 : 10 represents a 15 : 1 ratio !

Without entering further into the extensive data in my paper
(1914), it may be remarked that two things appear to be clearly
proven; (1) that Oe. mut. rubricalyx originated through a single
unit-change producing a new character-difference which is dominant ;
(2) that when the new form is crossed with a distinct species — Oe.
grandiflora — having a different physiology, the red-budded feature
comes out in F„ in varj'ing ratios in different families, 3:1, 4:1, 5:1,
and about 10 : 1. Not only is this the case, but occasionally inter-
mediate individuals as regards bud-pigmentation occur, and it has since
been found that these when selfed breed true to their intermediate

R. RuGGLBs Gates 359

condition, without reversion to either the ruhrinervis or the rubricalyx
type of pigmentation. Moreover, back-crosses of the original F^ hybrids
with rubricalyx intensify the pigmentation, while back-crossing with
[irandiflora dilutes and modifies it, producing a spotted condition of
the sepals in certain cases. I may perhaps be pardoned for main-
taining that all these facts confirm my view that, (1) the rubricalyx
pigmentation originated through a unit-change, as demonstrated by
the ratios before crossing and in the F^ hybrids; (2) this sharj)
unit-character may be modified and broken up by crossing with a
physiologicall\- diverse species. An adequate e.xplanation has not yet
been found for the F., ratios, such as .5:1 and 10:1, found in these
hybrids, but a little thought will show that any argument is fallacious
which attempts to argue backwards, as Shull has done, fi-om these ratios
to the original condition of the unit-character R before it was crossed. .

In Shull's paper he devotes considerable attention to the red
spotting of the rosette leaves in his hybrids. This is a conspicuous
feature of Oe. grandifiora, and I have devoted much time to the study
of its inheritance in hybrids with rubricalyx. There is an evanescent
stage in the development of the rosettes of these F^. hybrids, when on
superficial examination it seems easy to classify them into red-spotted
and non-red-spotted. But more intensive study shows that every
degree of spotting exists, from rosettes in which all the leaves are
well spotted to those in which only one or two tiny sjoots appear on
the whole rosette. In other words, there is a continuous series from
rosettes with a large amount of spotting to those with none at all.
This feature was not found to be correlated with any well-marked
condition of the adult plants (as is the case with the ventral red of
rubricalyx rosettes), and as the classification of the rosettes is neces-
sarily more or less arbitrary and hence not dependable, it was not
considered worth while publishing ratios in which there was such a
large margin of eiTor.

When Shull speaks of " negative correlation " between the pig-
mentation of the buds and of the stems and rosettes in his triple
hybrids, he is observing complex phenomena which have not been
analyzed. I have observed a large number of similar phenomena in
F.2, and F^ hybrids of grandiflora and rubricalyx, but have awaited
further data on the subject before publishing. It means, of course,
that red pigmentation in different parts of the same plant may be
separately inherited, as is known to be the case in various other plants.
Shull's " negative correlations " appear to depend, in part at least, upon

360 Origin and Behaviour of Oe.nothcra rubricalyx

the fact that (jrandifiora frequently has dark rod stems while in Oe.
Lamarckiana and its derivatives (except rubricalyx) the stems have
very little diffuse red, though they are covered with red papillae.
I have not, however, found this " negative correlation " to be uni-
versal, since for example in some families the liybrid plants have
red buds and red stems as well. It is probable that the alternation
in Shull's hybrids is really between the dark red stems of some
grandiflora races and the green or nearly gi-een stems of forms in
the Lmnarckiana series.

Finally, it must be made clear that ShuU's results, such as they are,
can only be used for reference if in every case, for ruhricalyx, we read
rnhricalyx x grandijiora.

LITERATURE CITED.

Gates, R. R. 1909. " An Analytical Key to some of the Segregates of Oenothera."
Ann. Rept Missouri Dot. Garden, Vol. xx. pj). 12.3 — 137.

1910. "The Material Basis of Mendelian Phenomena." Amer. X(tt., Vol.

XLIV. pp. 203—213.

1911. "Studies on the Variahility and Heritability of Pigmentation in

Oenothera." Zeitsckr. f. ind. Abst. u. Vera-b. Bd. iv. pp. 337 — 372, pi. 6, 5 figs.

1912. " Mutation.s in Plants." Bot. Jour. Vol. II. pp. 84—87, fig. 1.

1913. "A Contribution to a Knowledge of the Mutating Oenotheras."

Trans. Linn. Soc. London, Botany/, Vol. vill. pp. 1 — 67, pis. 1 — 6.

1914. "Breeding Experiments which show that HyliridiKition and Muta-
tion are Independent Phenomena." Zeitschr. f. ind. Abst. u. Vererh. Bd. xi.
pp. 209—279, 25 fig.s.

Shhll, G. H. 1914. "A Peculiar Negative Correlation in Oenothera Hybrids."
Journal of Genetics, Vol. IV. pp. 83 — 102, pis. 5, 6.

REGENT CHEMICAL INVESTIGATIONS OF THE
ANTHOCYAN PIGMENTS AND THEIR BEARING
UPON THE PRODUCTION OF THESE PIGMENTS
IN PLANTS.

By ARTHUR ERNEST EVEREST, M.Sc, Ph.D.
{Lecturer in Chemistry, University College, Reading).

The relationship— if any — which exists between the red, purple and
blue plant pigments (Anthocyans) and those of the yellow plant pig-
ments which are of the flavone or flavonol class is of considerable
interest to students of genetics.

Until quite recently all ideas upon this subject were based of neces-
sity upon evidence obtained from botanical investigation, as reliable
chemical investigation upon this subject was entirely lacking. At last
however definite chemical evidence bearing upon the relationship
existing between these two important classes of naturally occurring
pigments has been obtained, and in the present paper the author
summarises briefly the important points that have been investigated,
and points out their bearing upon the theories previously put forward.

Up to the present the consensus of opinion has been in favour of
the theory that the anthocyans are produced by oxidation of yellow
pigments of the Havone or flavonol series. The fact that these ideas
have failed to stand the test of chemical investigation — in every case
that has been examined in a chemically satisfactory manner — must now
be realised.

Attacking the problem with a view to establishing the chemical
nature and structure of the anthocyan molecule, Willstatter and Everest
(Liebig's Annalen, 1913, 401, 189) investigated a number of these pig-
ments, and in particular that of the corn-flower. Their results contain

362 Chemistry of Aiit/iocyati Plgmetitx in Plants

much that is of interest to those studying these pigments tiimi the
standpoint of genetics.

The corn-flower pigment, which was obtained pure and cr3'stailine,
was proved to be a glucoside, which on hydrolysis with acids yielded
two molecules of glucose and one molecule of the true pigment — a fine
crystalline substance — and it was also shown that in all the cases
examined the pigments occurred in the jjlants only as glucosides. These
results have been fully confirmed by later work, and the idea, so fre-
quently put forward, that the pigments occur together in plants both as
glucosides and non-glucosidcs, must as a result be abandoned. In cases
where previous workers obtained mixtures of glucoside and non-glucoside,
e.g. Heise, Glan, it was due to partial hydrolysis during the processes of
isolation. This generalisation simplifies somewhat the problems to be
dealt with when the processes going on within the plant tissues come
to be considered, but by no means so much as the proof that the blue
anthocyan pigments are alkali salts of compounds which in the free
state are violet or violet-red in colour, and which are capable of uniting
with acids to form oxonium salts which are red in colour. Beyond these
results the same authors demonstrated that, at least in the case of the
corn-fiower pigment, the decolourisation which so readily takes place in
aqueous solution was not due to reduction as has been assumed so
frequently. It was further shown that by oxidation the pigment of the
corn-flower readily passed to a yellow pigment which had properties
similar to those of the fiavone or flavonol derivatives.

This work brought the present author to the opinion that the
anthocyans were reduction products of the fiavone or flavonol com-
pounds, whereas Willstatter favoured rather the idea that they were
related to some unknown flavone or flavonol derivatives in which a
hydroxyl group occurred in the benzopyrone nucleus in the para
position to the linkage of the oxygen atom of the pyrone ring. The
relationship is clearly shown by the formulae I and II, and it will be
seen that this makes the anthocyan an oxidation product of the com-
pound II.

OH

■Ch

HO-

MO

I. Antliocyau. II. Flavone derivative.

A. E. Everest 363

The present author's idea that the anthocyans were reduction
products of flavone derivatives was deepened by consideration of the
work of Keeble, Armstrong and Jones {Roy. Soc. Pruc. 1918, B, 113).

Working upon these ideas he carried out exjjeriinents that clearly
showed that by the careful reduction of flavone derivatives a series of
red pigments having the properties of anthocyanidins could readily be
obtained ; moreover, contrary to the almost generally accepted idea, he
showed that quite similarly glucosides of the flavone series yielded —
without intermediate hydrolysis — glucoside red pigments in every way
similar to anthocyanins. From these investigations the present author
put forward the scheme :

OH
VOH HOY

H " reauchon H O H +Tm5

III. Flavone derivative. IV. Colourless or faintly V. Anthocyan pigment,

coloured intermediate product.

to represent the change from flavone derivative to anthocyan, and the
formula V as that of a typical pigment of the latter group (Everest,
Roy. Soc. Proc. 1914, B, 444). As further support for these conclusions
the work of Watson and Sen (J. Chem. Soc. 1914. 389) and Combes
{ComjJt. Rend. 1913, 157, 1003) were cited.

Shortly after the appearance of the above mentioned paper by the
present author, Willstatter and his collaborators published a short but
important paper (Sitzher. k. Akad. Wiss., Berlin, 1914, xil. 402) describing
the continuation of the work on the isolation of pure anthocyans from
flowers and fruits. They had obtained in a chemically pure and crystal-
line condition the anthocyan pigments from no fewer than nine flowers
or fruits, all of which pigments proved to be glucosides, and by hydrolysis
of the glucosides obtained the colour components also chemically pure
and crystalline. They described the decomposition of the non-glucoside
pigments (anthocyanidins) by the action of warm alkalis, and the products
produced thereby. As the result of their work they put forward for the
anthocyan molecule a formula identical with that previously proposed
by the present author. They pointed out however that their results
did not allow them to finally decide between it (V) and VI. They
further concluded that the scheme previously put forward by the
present author (see above) should represent the passage of a flavonol
derivative to an anthocyan pigment, and additional support to this was

364 Chemistry of Anthocycui Piymeiits in Plants

brought by their showing that the decolourisalioii of anthocyans in
aqueous solution was produced by the addition of a molecule of water

HO

VI.
to the anthocyan molecule, the addition causing loss of a molecule of
hydrochloric acid ; addition of hydrochloric acid to the decolourised
product causes reversal of these changes, thus :

■^'i -<^« -^ -o/Y'°-S ^IJJ- -^ -o^-,

i^A t"" °" • — \XJ-o„^-o' ^- \XJ-'" -<'"

■q -< V-OH

o o

H M

Despite this they criticist-d the present author's conclusious that the
pigments obtained by hiiu by the reduction of flavone derivatives and
their glucosides were true anthocyanidins and anthocyanins. They
stated that such pigments were soluble in ether, and also differed in
stability from the natural anthocyans.

To this criticism, Whcldale and Bassett {Journal of Genetics, 1914, iv.
No. 1, p. 103) added their support, but in a paper recently read before
the Royal Society (forwarded for publication July 15, read Nov. 12) and
in a note in the Journal of Genetics (1914, iv. No. 2, p. 191^ the
present author has shown that these criticisms are not well founded.

Since these papers were sent for publication, a further paper by
Willstittter and collaborators has come to hand (Sitzber. k. Akad. Wiss.,
Berlin, 1914, 769), in which, after repetition of the present author's
experiments, they withdraw their criticism of his conclusions, for they
find indeed that by reduction Quercetin really does produce the
anthocyan pigment Cyanidin. Their experiments show that whilst at
temperatures below 0°C., the hydrochloride of a new substance — which
they term Allocyanidin — is produced, at higher temperatures the product
of reduction of quercetin consists of a mixture of allocyanidin chloride
and cyanidin chloride, and moreover they were able to show that the

A. E. Everest

365

latter is in every way identical with the natural cyanidin chloride
obtained from the com-flower or rose.

Thus the structure of all the pigments of this group at present
known in a pure condition becomes evident, and the manner in which
they are related to naturally occurring flavone derivatives is clearly
shown by the following formulae :

HO

Flavone derivative.
O

HOy

Kaempferol.

Quercetin.

OH

Anthocyan.
CI

HO

OH

Pelargonidin chloride.

HO-i?

Cyanidin chloride.

HO

Myricetin.

HO-

Delphiuidiu chloride.

Myrtillidin chloride has one, Oenidin chloride two methoxy groups
in place of hydroxy groups in Delphinidin chloride, but the position
of these groups is, as yet, uncertain ; Willstatter tentatively gives
them as:

C H,o-

OH

0CH3
OH

Myrtillidin chloride.

Oenidin chloride.

3(56 Cheinistry of Anthocyaii Pigments in Plants

It will bo noticed that all these are derivatives of flavonol VI and
thus tar no anthocyans corresponding to flavone itself VII have been

C-OH

c

II
o

VI.

II
o

VII.

C— H

obtained from natural sources. As the pi-esent author has already
pointed out in previous publications, in that flavones occur naturally
and on reduction yield red pigments, doubtless anthocyans related to
flavones will be found as the result of further investigation of the
naturally occurring pigments.

The anthocyan pigments thus far isolated in a chemically plire and
crystalline condition, and whose structures have now been definitely
established, are :

(1) Cyanin, obtained from the corn-flower, and identical with that
obtained from the rose (gallica). A glucoside which on hydrolysis
yields the non-glucoside pigment cyanidin and two molecules of
glucose.

(2) Pigment of the cranberry, a mono-saccharide of cyanidin, wiiich
on hydrolysis yields cyanidin and one molecule of galactose.

(3) Pelargonin, obtained froin Pelargonium zonale, yields on
hydrolysis pelargonidin and two molecules of glucose.

(4) Oenin, from deep-coloured wine grapes, gives oenidin and one
molecule of glucose.

(.5) Delphinin, from larkspur (pur])lt'), yields delphinidin, two
molecules of glucose and two molecules of ^j-oxybenzoic acid.

(6) Myrtillin, from bilberry, gives myrtillidin and one molecule of
glucose.

(7) Pigments from two types of hollyhock, one of which j-iolds one
miilecule, the other two molecules of glucose and the non-glucoside
pigment myrtillidin.

The chemical investigatinns above mentioned having established
the relationship which the anthocyans bear to the flavone derivatives,
the fact that the distribution of oxidases and pei'oxidases in the plant
in many cases coincides with the ilistribution of the anthocyan pigments

A. E. Everest 367

can no longer be accepted as favouring the suggestion that the antho-
cyans are oxidation products of the fiavones; its true interpretation has
yet to be sought.

Chemical evidence has now established the facts :

(1) That the anthocyans always occur asglucosides (anthocyanins).

(2) That the same pigment may be capable of showing a blue,
purple or red colour, according as it exists as alkali salt, free pigment
or oxonium salt of some acid. All anthocyans do not, however, form
blue alkali salt.s.

(3) That the anthocyans may be obtained from flavonols by reduc-
tion followed by spontaneous dehydration as shown above.

(4) That glucosides of flavonols can pass, by reduction, toglucoside
anthocyans (anthocyanins) without intermediate hydrolysis.

The points (3) and (4) doubtless apply also to flavones, but as no
natural anthocyans related to these have, as yet, been isolated, proof of
this is naturally unavailable.

(5) All analytical evidence points to the molecular weights of the
anthocyanidins being of the order of those of the flavonols.

The author does not propose to go deeply into the Mendelian
significance of the chemical results discussed above, but would suggest
that they appear to show that the factors R and B so frequently used
to represent the power to produce red and blue anthocyan pigment
respectively, are really complex factors representing power to produce
different conditions of acidity and alkalinity in the cell sap, together
with the power to produce anthocyan pigment independent of whether
it is in the red, purple or blue form. It ought also to be noted that
the factors R and B if looked upon thus must affect the production of
ivory and yellow, for it is well known that in many ivory flowers the
pigment of the flavone series is present, and it is only necessary to
make the cell sap alkaline in order to produce a fine yellow flower.
These are however matters that are better left for those researching
upon ISIendelian problems to deal with, but now that chemical investi-
gation has thus far cleared the ground, the problems involved in the
production of these pigments become somewhat more clear, and research
in this field of botanical work should be stimulated and helped thereby.

Journ. of Gen. iv 24

OUR PRESENT KNOWLEDGE OF THE CHEMISTRY
OF THE MENDELIAN FACTORS FOR FLOWER-
COLOUR.

PART II.

By M. WHELDALE,

Fellow of Newnham College, Cambridge, and formerhj Research Student
of the John Innes Horticultural Institution, Merton, Surrey.

Since the appearance of Part I of the author's paper on this subject^
further work on anthocyanin has been published by Willstatter, in
conjunction with Bolton, Nolan, Mallison, Martin, Mieg and Zollinger.
The present paper is concerned with the bearing of these later results
on the genetics of flower-colour.

In Part I of the author's paper reference was made to Willstatter 's
first publication on anthocyanin" and the views contained therein,
namely, that anthocyanin is an oxonium compound, which is purple in

1 Wheldale, M., "Our Present Knowledge of the Chemistry of the Mendelian Factors
for Flower-colour." Journal of Genetics, Cambridge, 1914, Vol. iv. (No. 2), p. 109. This
communication was delayed six months in the press, and at the time of writing, Willstatter's
second communication was not available and his third communication was not yet
published.

The question as to the identity of the red products, formed artificially by reduction
from flavones, with natural anthocyanins is considered in a separate paper in this journal : —
Wheldale, M., and Bassett, H. LI., " On a supposed Synthesis of Anthocyanin." Journal
of Genetics, Cambridge, 1914, Vol. iv. (No. 1), p. 103. Owing to the above-mentioned
delay, the latter paper appeared first and as regards this particular question of identity
expresses the author's more recent views.

- Willstatter's communications referred to are : — (1) Willstatter, E., und Everest, A. E.,
"Ueber den Farbstoff der Kornblume." Liehigs Ann. Cliem., Leipzig, 1913, Bd. 401,
p. 189. (2) Willstatter, E., " Ueber die Farbstoffe der Bliiteu und Friichte." SitzBer.
Ah. Wiss., Berlin, March 1914, p. 402. (3) Willstatter, E. , und Mallison, H., "Ueber
die Verwandtschaft der Anthocyane und Flavone." SitzBer. Ak. Wiss., Berlin, July 1914,
p. 769.

370 Chemist nj of Menclelian Factors Jor Flotver-Colour

its neutral state and forms red oxonium salts with acids and blue salts
with alkalies.

Willstatter's second and third papers deal with the preparation and
constitution of a number of anthocyanins in addition to the pigment of
the Cornflower. The main facts of importance in both communications
will be set out first as follows.

The analyses, published in Willstatter's first paper, of the cyani<lin
of the Cornflower in the form of the chloride have been found by him
to be incorrect owing to imperfect drying and a new foi-mula f'loni
analyses (by combustion) is given as :

Cyanidin chloride, CbHioOsHCI (original formula CifiHuOyHCl).

The anthocyanin of the Rose (Rosa gallica) was found to be a
diglucoside of cyanidin and also the pigment, idain, from the Cranberry
to be a galactoside of cyanidin, giving two molecules of galactose on
hydrolysis.

The additional anthocyanins' isolated are:

Delphinin (from Delphimvm flowers), which gives on hydrolysis
2 mols. of glucose, 2 mols. of ^-oxybenzoic acid and 1 mol. of delphinidin,
C^H^AHCl.

Pelargonin (from Pelargonium flowers), which gives on hydrolysis
2 mols. of glucose and 1 mol. pelargonidin, CisHioOsHCl.

Onin (fi-om Grapes), which gives on hydrolysis 1 mol. glucose and
1 mol. onidin, C,,H„0;HC1.

Myrtillin (from Bilberry fruits and flowers of Hollyhock, Althaea
rosea), which gives on hydrolysis myrtillidin, Ci6H].,0;HCl and glucose.

Malvin (fi-om Mallow flowers), which gives on hydrolysis malvidin,
CHiAHCl.

All the above substances were prepared in the ciystalline form as
hydrochloric acid salts and- apparently analyses were madi^ from such
salts only.

The formulae for the pigments free fi-om hydrochlcjric acid may be
compared with the flavones as follows :

Cyanidin, CisHjoOg, isomeric with luteolin, kampherni and flsetin.

Pelargonidin, CijHioOs, isomeric with apigenin.

Delphinidin, CisHioO., isomeric with quercetin and moriii.

Onidin, C,.H,oO,(CH.,)3 ] .u w . , ■ t- f i i .•

• ,,•,• ^ TT ^ ./~iTT N are methylated derivatives ot ilclplu-
Myrtilhdin, CsHioO^ (CHj) y . ^ '

Malvidin C,5H,oO,(CH,)„ J °

' In the present paper the distinction made by Willstatter between anthocyanin
(glucosidal product) and anthooyanidin (non-glucosidal product) is not employed.

M. Wheldale

371

When heated with alkali, the various pigments split up, as in the
case of flavones, into a phenol and an oxybenzoic acid, which were
identified in each case :

Cyanidin gave phloroglucin and protocatechuic acid.

Pelargonidin „ „ „ jo-oxybenzoic

Delphinidin „ „ „ gallic acid (not isolated in pure

state).

On the basis of the above decompositions and analyses (by combus-
tion) Willstatter first suggests the following alternative constitutional
formulae for three of the pigment salts :

OCl

v/\/

HO

OH

OH

or

HO

OCl

HO T

OH

Cvanidin chloride.

\/0" II.
OH

OCl

HO

or

OCl
HoXV^

HO

OH

II.

OH

Pelargonidin chloride.

HO

y\

OCl

(A,

r<

OH

^OH
^H

or

HO

r^

OCl

HO

OH

HO

X

OH

Delphinidin

chloride.

HO

OH

°" II

24—3

372 Chemistrji of MendeUau Factors for Flower-Colonr

In water and alcohol solution both the anthocyanins and their salts
tend to become colourless with the formation of a colourless iso-fonn.
Willstatter states that the analysis of the colourless iso-form in the
case of delphinidin has shown that the change from the quinonoid to
the non-quinonoid combination takes place with the addition of a
molecule of water, though no more light is thrown on the reaction than
is given in the following equation :

Ci^H.oCHCl + H,0 = HCl + C,.,H,.A.

In acid solution, on the other hand, and in the solutions of some
neutral salts', the anthocyanins are stable owing to the formation of
the oxonium salts, and the stability is maintained in the absence of
excess of water.

Formulae of the type II, as shown above, are discarded by Will-
stiitter since, for example, if such a formula were correct for cyanidin,
it should be possible to obtain maclurin as an intermediate product of
decomposition and this he did not achieve.

Adopting therefore formulae of type I, Willstatter then points out
that the relationship between anthocyanins and Havones is such that
it should be possible to obtain the red pigments from the yellow by
reduction and, converselj-, the yellow from the red by oxidation.

The question as to the formation of anthocyanins from flavones on
reduction by means of nascent hydrogen has been considered in the
author's previous paper. The phenomenon was first investigated by
Combes- who maintains that he obtained a cr3'stalline red pigment
identical with natural anthocyanin by reduction of a naturally occurring
flavone : these statements, however, are not supported bj' analyses.
The same view of the identity of products of flavones with natural
anthocyanins has been held by Everest''.

Willstatter, also, has investigated the reduction products of flavones
in view of his hypothesis as to the origin of anthocyanins and he claims
to have shown that in the process two products are formed, of which
one is a true anthocyanin, the other not. In his experiments, an
alcoholic solution of quercetin acidified with hydrochloric acid is reduced
with sodium amalgam or magnesium. The bulk of the purplish-red

1 See Willstatter's first paper.

- Combes, E., C. It. Acad. Sci. , Pans, 1913, T. cLvn. p. 1002 and p. 1454, and T. cLviti.
p. 272.

3 Everest, A. E., "The Production of Anthocyanins and Anthocyanidins." Proc.
Roy. Soc, London, 1914, B, Vol. Lxxxvii. p. 444. Ibid. Pt II. 1915, B, Vol. r.xxxvm.
p. 326.

M. Wheldale

373

product formed he terms allocyanidin and he suggests that the reaction
proceeds as follows with the opening of the pyrone ring :

HOi'^Y^ <( )>0H HO

"°? t2H„

PH ^ OH /H

OH °^^/C

H„0

Allocyanidin forms a crystalline compound with hydrochloric acid
and it is apparently on the analysis (by combustion) of this compound
that Willstatter bases the above constitution of allocyanidin.

Allocyanidin chloride is unstable on heating in dilute hydrochloric
acid but when the crude solution from reduction of quercetin with
nascent hydrogen is diluted and heated in order to decompose allo-
cyanidin chloride the solution does not entirely lose colour owing to
the presence, according to Willstatter, of a small amount of a second
substance. The latter formed with hydrochloric acid a crystalline
product which on isolation and analysis proved to be identical with
cyanidin chloride. From 33 gms. of quercetin, 0"165 gm. only of the
product was obtained. The identity was established on the results of
analysis (by combustion), appearance, solubilities and properties in
general. It is suggested by Willstatter that the reaction proceeds as
follows :

HO

+-H.,0

Since the constitution of quercetin is known, Willstatter considers
the formula for cyanidin chloride to be established by the above reaction
and very probably also the formulae for delphinidin and pelargonidin
clilorides :

OCl OH

OCl

-°fY]-< >OH

OCl OH
HO^^^X / -\oH
^ X>H

\A/OH

HO C
H

HO C

H

HO C
H

Cyanidin chloride.

Pelargonidin chloride.

Delphinidin chloride.

374 Chemistry of Meudelian Factors for Floicer-Colour

Other anthocyanins are represented as methyl deri\atives of
delphinidin :

OCl OH ^°C' OH OCl OH

\ HO/VS
/OH
OH

Myrtillidin chloride. Onidin chloride.

He points out moreover that the anthocyanidins in the acid-free
form may be regarded as forming a series of reduced Havones :

Luteolin, kampherol, and

fisetin CsHjoOs — *- CisHijOj pelargonidiii.
Quercetin CisHjoO^ — »■ C,5Hi„0s cyanidin.
Myricetin CjsHjoOg — *- CisHinO, delphinidin.

Bringing Willstatter's views to bear upon the Mendelian factors for
flower-colour, we necessarily come to the following interpretations. The
chromogens of the pigments are flavones and the factor for colour is
the power to bring about a simple reduction of the flavone accompanied
by a change from divalent to tetravalent oxygen and a pyrone to a
quinonoid structure. If the cell-i5ap is neutral the anthocyanin has the
structure of an inner oxonium salt and is purple. A reddening factor
must be interpreted as one which produces an acid cell-sap whereby
the anthocyanin is enabled to form a red acid oxonium salt. Similarly
a bluing factor is one which produces an alkaline cell-sap, blue antho-
cyanin being an alkaline salt of the purple neutral compound.

With the exception of the view that the anthocj'anins are derived
from flavones which was first suggested in 1909', the author's results
are not in agreement with those of Willstatter. In the case of
Antirrhinuin investigated by the author in conjunction with Ba-ssett,
there is no doubt that the chromogen is the flavone, apigenin. On
Willstatter's hypothesis that the anthocyanins are reduced flavones, the
formula for Antirrhinum anthocyanin should be CijHioOj (apigenin
being C15H10O5), giving the percentage composition :

C ... 70-86%,
H ... 3-94 7,,
O ... 2.5-20 7„

' Wheldale, M., "On the Nature of Anthocyanin." Proc. Phil. Soc, Cambridge,
1909, Vol. XV. p. 137.

M. WlIELDALK 375

whereas the percentage compositions actually found for the two forms
of Anth-rhinum anthocyanin are :

Red Magenta

C ... 51-81% C ... 50-50%

H ... 5-01 7^ H ... 511 %

O ... 43-18% O ... 44-39 7„

Moreover the artificial red product prepared from apigenin by
reduction with sodium amalgam does not give the qualitative reactions
nor has it the percentage composition of the natural anthocyanin.

The red and magenta anthocyanins from Antirrhinum were not
obtained in crystalline form, nevertheless their purity was guaranteed
by the concordance of analysis results when the pigments were prepared
independently from entirely different varieties. Also the products
analysed were the pigments themselves and not the hydrochloric acid
salts.

The analyses show that both red and magenta contain more oxygen
than apigenin. Moreover the red pigment is not an acid salt of the
purple or magenta : both are precipitated from acid solutions. Nor is
the magenta an alkaline salt of the red. They are different substances,
and cannot be converted the one into the other by such means.

In the work on Antirrhinum, the problem which has been before
the author may be exjjressed thus : — What are the actual processes
which lead to anthocyanin formation in the living cell ? Only the
answer to this question will enable us to interpret the Mendelian
factors correctly.

Willstatter's percentage formulae for several anthocyanins coupled
with the production, from quercetin by reduction, of a very small
quantity of a substance claimed to be identical with cyanidin are not
convincing reasons for regarding the natural process of pigment for-
mation as one of reduction. In this respect the important point is
whether cyanidin is formed from tfuercetin, delphinidin fi-om myricetin,
etc., in the living plant. Willstatter makes no mention of the flavones
accompanying the anthocyanins he has isolated. Myricetin which
should be the chromogen of delphinidin is at present known in Myrica,
Rhus, Haematoxylon and Arctostaphylos. From Delphinium consolida
kampherol has been isolated but it is quite possible that other flavones
may be present in addition or different flavones in other species.

Further evidence is still needed of the connection between antho-
cyanin and flavone in the same plant, and between the natural

o7H Chemistrif of Menddian Factors for Floiver -Colour

anthocyanin and the artificial pigment prepared from the same Havone.
Only in Antirrhinum are such relationships knuwn and they arc not so
far in accordance with the reduction hypothesis.

Willstjitter dismisses as disproved the hypothesis suggested by the
author" some years ago, with a view to explaining some of the phe-
nomena, both physiological and chemical, of anthocyanin production.
The hypothesis supposes several of the hydroxyls of the flavones in tlie
living cell to be replaced by sugar and that only after hydrolysis of
certain hydroxyls can changes take place at these points with the
production of pigment. The hypothesis is more within the province of
plant physiology than chemistry and was the outcome of observations
upon the distribution of pigment in the tissues and the effect of factors
such as light, temperature, drought, injury, sugar feeding, etc., on
anthocyanin formation. The hypothesis has been said to be rendered
valueless by the fact that in the formation of artificial anthocyanin,
mono- or even di-glucosides of flavones can be employed and the pig-
ment is formed on reduction in the cold without hydrolysis.

The existence of stable di-glucosides of quercetin which can be
isolated and which, when treated in hot acidified alcoholic solution
with nascent hydrogen, give a red product is no criterion of the con-
ditions in which the quercetin exists in the living cell, nor of the
reactions which convert it into anthocyanin.

Note. I should like to take this opportunity of correcting two errors
made by me in a previous paper written jointly with Mr Bassett, i.e. "On
a supposed Synthesis of Anthocyanin " and published in this Journal,
Vol. IV. No. 1, p. 103.

On page 104, line 3, of the above paper, read :

" glucoside (of flavone) -I- water '*~7 chromogen (flavone)

+ sugar X (flavone) + oxygen = anthocyanin."

On Jjage 105, line 9, read :

" then reducing without removal of sugars."

' Wheldale, M., " On the Formation of Anthocyanin." Journal of Genetics, Cambridge,
1911, Vol. I. p. 133.

CAMBRIDGE: PRINTBD BY JOHN CLAT, U.A. AT THE UNIVEBSITY PRESS

376 Chemistry of Menddian Factors /or Flower-Colour

anthocyanin and the artificial pig^nent prepared timn the sune tiavone.
Only in Antirrhinum are such relationships known and they are not so
far in accordance with the reduction hypothesis.

Willstiitter dismisses as disproved the hypothesis suggested by the
author' some years ago, with a view to explaining some of the phe-
nomena, both physiological and chemical, of anthncyanin production.
The hypothesis supposes several of the hydroxyls of the flavones in the
living cell to be replaced by sugar and that only after hydrolysis of
certain hydroxyls can changes take place at these points with the
production of pigment. The hypothesis is more within the province of
plant physiology than chemistry and was the outcome of observations
upon the distribution of pigment in the tissues and the effect of factors
such as light, temperature, drought, injury, sugar feeding, etc., on
anthocyanin formation. The hypothesis has been said to be rendered
valueless by the fact that in the formation of artificial anthocyanin,
mono- or even di-gluco.sides of flavones can be employed and the pig-
ment is formed on reduction in the cold without hydrolysis.

The existence of stable di-glucosides of quercetin which can be
isolated and which, when treated in hot acidified alcoholic solution
with nascent hydrogen, give a red product is no criterion of the con-
ditions in which the quercetin exists in the living cell, nor of the
reactions which convert it into anthocyanin.

Oiving to an error in alignment it is requented that this slip be
substituted fur the Note on p. 376 of Vol. IV.

Note. I should like to take this opportunity of correcting two errors
made by me in a previous paper written jointly with Mr Bassett, i.e. " On
a supposed Synthesis of Anthocyanin " and published in this Journal,
Vol. IV. No. 1, p. 103.

On page 104, line 3, of the above paper, read :
" glucoside (of flavone) -l- water ±:^ chromogen (flavone) + sugar
X (flavone) + oxygen = anthocyanin."

Ou page 105, line 9, read :

" then reducing without removal of sugars."

cambkidge: printed by john clay, m.a. at the dniversity press

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This \olume is believed to be the first attempt, in any language, to
cover the whole field of investigation. It is based throughout upon the
manuscripts, and the collection and classification of the materials has been
rhe work of several years.

Part I deals with the Greek and Eastern Canticles under the headings
of Greek, Graeco-Latin, Arabic, Armenian, Coptic, Ethiopic, Georgian,
Persian, Slavonic, and Syriac. Part II with Latin and Western Canticles.

Reproductions of three MSB are included in the book, together with
Indexes of Canticles and of MSB cited.

Nestorius and his place in the History of Christian Doctrine. By Friedrich
hoofs., D.D., Phil.D., Professor of Church History in the University of
Halle- IVittenherg., Germany.

Crown Svo. pp. viii + 132. Price 3^. 6J. net

The book consists of four lectures which tiie author was invited to give
last year at London University ; they are entitled: I. A Fresh Interest in
Nestorius Recently Awakened. II. The Tragedy of Nestorius' Life.
III. The Doctrine of Nestorius. IV. Nestorius' place in the History
of Christian Doctrine.

"The recent discovery of Nestorius' Liber HeraciiJis has awakened a new interest in
a theologian not hitherto much regarded by modern historians, and for orthodox
churchmen condemned as a heretic who saw in Christ no more than an inspired man.
Dr Loofs, in his learned and well-reasoned study, sets out to show that this is not
a tenable theorv of the doctrine of Nestorius." — The Scotsman

THE MODERN BIBLE— THE POEM OF JOB

The Bible of To-Day. By the Rev. Alban Blakiston., M.A.
Large Crown 8vo. pp. xvi + 240. Price JJ. net

In this book an attempt is made "to introduce the student of the Bible
to what is known as the historical, or critical, method of studying the
Scriptures and investigating their messages. The volume does not aim at

supplying introductions to the separate books The purpose is, rather, to

present to the reader the point of view which is responsible for applying the
historical method of treatment to the sacred pages, and to envisage the
'atmosphere,' intellectual and religious, which is the outcome of that
treatment

In an introductory chapter the question of Inspiration is briefly
discussed. The second chapter deals with the Text of the Old Testa-
ment, and seeks to trace, in outline, the history of its different writings;
endeavouring to grasp, on the one hand, what were the causes which
determined their form and contents ; and, on the other hand, how they
came to be combined in a single volume to which a peculiar sanctity
was attached. The third chapter deals with the New Testament upon
similar lines. And, in the last chapter, it is attempted to show what were
the influences which contributed to the development of the Jewish and
Christian religions, in so far as the books of the Old and New Testaments
exhibit this sort of dependence. Incidentally a great many other subjects
are touched upon."

The Poem of Job, translated in the metre of the Original. By Edward G.
King, D.D., Sidney Sussex College, Cambridge.

Pott +to. Paper boards, parchment back. pp. xii-|-ii6. Price 5J. net

The system of translating Hebrew poetry according to the principle of
accented syllables the author has previously explained in his volume on
Early Religious Poetry of the Hebrews in the series of Cambridge
Manuals. " It appears to me," he says, " that the English language well
lends itself to this rhythm, and that much of the beauty of our Bible
Version is due to the fact that the translators, from time to time, fall into
it, all unconsciously; e.g. Job iii. iq :

'The small and great are there ;
And the servant is free from his master.'

Here, as in Hebrew, the rhythm depends not on the number of syllables
but on the beat of the accent."

" Dr King's book will be valued by all who want to read the great dramatic poem

from end to end at one sitting, as it ought to be read It should receive a cordial

welcome from all who can appreciate a really useful and illuminating rendering ot the
great dramatic poem ot which Carlyle said, ' I call it, apart from all theories about it,
one of the grandest things ever written with pen.'" — The AherJeen Journal

5

THE CAMBRIDGE PUBLIC HEALTH SERIES

Isolation Hospitals. By H. Franklin Parsons, M.D. (Land.), D.P.H.
(Carnb.). Formerly First yfssistant Medical Officer of the Local
Government Board.
Demy 8vo. pp. xiv + 276. With 55 text-figures. Price 12J. 6J. net

111 writing this book the special aim of the author has been to produce
a work which shall be of practical use to members of local authorities,
medical officers of health, hospital superintendents and others interested

A

gMiiiJiJiiiJiiiii

Fig. 29. Elevation of part of a sanatorium pavilion of Doecker construction
for 20 beds. The administration buildings are not shown

in the establishment or management of hospitals for infectious diseases ;
and he hopes that the experience gained during a long official career may
have enabled him to include some useful information and suggestions not
otherwise readily obtainable. — Extract from the Preface

The Bacteriological Examination of Food and Water. By JVilliam G.
Savage, B.Sc, M.D. (Land.), D.P.H. County Medical Officer of

Health, Somerset. Late Lecturer on Bac-
teriology, University College, Cardiff".

Demy Svo. pp. x+174. With 16
illustrations. Price ys. dd. net

The author feels that existing text-
books upon Bacteriology, lucid and
complete as they are, are concerned
chiefly with the pathological aspect ;
whereas the treatment given to the
bacteriological examination of water,
air, foods, and the like is commonly
inadequate. " The aim of this volume
is to remedy this defect and to make
available a practical manual dealing not
only with the examination of these
substances but also with the deductions
to be drawn from the bacteriological
data obtained from their examination.
Much of the available information
is only at present to be found in
original papers not always readily ac-
cessible."
6

10. B. euteritidis sporogencs
enumeration jar

DRYDEN— ROMAN LAW

Lectures on Dryden. Delivered hy A. IV. Ferrall, Litt.D., King Edivara
Vll Professor of English Literature and Felloiv of Trinity College,
Cambridge. Edited hy Margaret De G. Verrall.

Demy Svo. pp. viii + zya. Price yj. (ni. net

"In October and November 191 1, Dr Verrall, as King Edward VII
Professor of English Literature, delivered a course of twelve lectures on
Dryden, thus carrying out the intention which he formed as soon as he
was appointed to the English Chair. The reason for the selection of this
subject was not only his own long-standing admiration for Dryden, but
the importance of his work in the development of English prose and verse,
and its comparative neglect among the younger lovers of literature at the
present day The present volume reproduces strictly the original manu-
script notes as arranged for delivery." — Preface

Contents : I. Dryden's Work, Character and Influence. II. The Epiules.
III. Absalom and Achitophel. IV. The Quatrain Poems; Stanzas on Cromnxel/
and Annus Mirabilis. V. Literary Criticism in the Age of Dryden ; the 'Unities' ;
the Essay of Dramatic Poesy. VI. The Religious Poems; Religio Laid and The
Hind and the Panther. VII. The Development of the English Ode ; Dryden's
Influence on Lyric Poetry. VIII. The State oj Innocence ; Dryden and Milton.
IX. All Jor Lo've ; or the World Well Lost. Index.

"Verrall's passion for literature as a living thing, his detestation of pedantry of all
kinds, his love of the parado.x that stimulates thought... all these things combined with
his great power of lucid exposition and his almost uncanny gift of declamation to make
it impossible that he should fail." — The Morning Post

Hisiory of Rotnan Private Laiv. Part IL Jurisprudence. By E. C.

Clark., LL.D.., soynetime Regius Professor of Civil Law in the University
of Cambridge, also of Lincoln s Inn, Barrnter-at-Law.

Crown Svo. In two volumes. Vol. I, pp. xiv-l-432. Vol. II, pp. iv-l-370.

Price 2!i. net

"This work," says the author in his Preface, "was written as part of
a History of Roman Private Law, which I can scjircely hope to complete,
but for which I have been collecting notes and other materials during
many years. It is therefore intended primarily for readers who take the
study of Roman Law with Jurisprudence, and necessarily involves some
considerable acquaintance with the former subject. It does not affect to
be a Manual, being intended rather for students than candidates for
examination."

The Glasgow Herald describes the book as an " elaborate, erudite, and
leisurely examination of the nature of jurisprudence under constant refer-
ence to the Roman Law and the English institutional writers, at once
analytical, descriptive, and comparative. If it has little of the formal
precision of Holland's well-known text-book, its discursive method admits
of wise and full discussion of such problems as the relation of moralit)
to law, and of law to the state, which are of immediate interest to man]
other persons than students."

7

GREEK SCULPTURE AND MODERN ART

Greek Sculpture and Modern Art. By Sir Charles IValdstein^ Litt.D.,
Ph.D., L.H.D. Fellow and Lecturer of King's College, Cambridge.
Sometime Slade Professor of Fine Art, Reader in Classical Archaeologx,
Director of the Fitzwilliam Museum, Cambridge, and Director of the
American School of Archaeology, Athens.

Demy Svo. pp. xii + 70. With 7S plates. Price js. 6J. net

The book comprises two lectures, delivered by the author in Februar\-
1 91 3 to the students of the Royal Academy Art School ; these are now

published in a more permanent
form, as it is thought that the)-
will prove useful, not only to
students of art, but also to the
general public, as an introduction
to the study of sculpture. The
author's aim has been to show that
" whatever justification there be
in the new aspirations, in the
new methods, and in the new
outlook, the study of Greek
sculpture still remains, and will
always remain, as far as its
fundamental principles and its
main achievements are con-
cerned, a subject which you
can study with profit and at
some stage you must study."

The author's allusions to the
famous examples of both ancient
and modern art are illustrated
by a very large number of pho-
tographs. " These," says The
Times, " are judiciously selected,
and the text supplied is helpful ;
...the book will be a welcome
additioxi to the libraries of those
to whom Greek art is a source
of undying joy."

As an Appendix, Sir Charles
Waldstein reprints a leading
article which appeared in The
Times on the subject of his lec-
tures, and his own reply to some
Le Penseur by Rodin criticisms which it contained.

''^:i'

DYNAMICS - P HOTO-ELECTRICITY

Dynamics. By Horace Lamb^ Sc.D., LL.D., F.R.S. Formerly Fellow of
Trinity College, Cambridge. Professor of Mathematics in the Victoria
University of Manchester.

Demy 8vo. pp. xii+344. Price loi. (sd. net

In dealing with his subject the author, avoiding the abstract treatment,
which he regards as " likely to bewilder rather than to assist the student,"
has preferred to follow the method adopted by Maxwell in his Matter and
Motion. Some account of the more abstract, if more logical, way of
looking at dynamical questions is given at the end of the book. The
author has been at pains to collect useful examples for practice, preference
having been given to those which are simple rather than elaborate from
the analytical point of view.

Contents, — I. Kinematics of Rectilinear Motion. II. Dynamics of Rectilinear
Motion. III. Two-Dimensional Kinematics. IV. Dynamics of a Particle in Two
Dimensions. Cartesian Co-ordinates. V. Tangential and Normal Accelerations.
Constrained Motion. VI. Motion of a Pair of Particles. VII. Dynamics of a System
of Particles. VIII. Dynamics of Rigid Bodies. Rotation about a Fixed Axis.
IX. Do. Motion in Two Dimensions. X. Law of Gravitation. XI. Central Forces.
XII. Dissipative Forces. XIII. Systems of Two Degrees of Freedom. Appendix.
Note on Dynamical Principles. Index.

"Professor Lamb has now fulfilled the promise made about a year ago to issue the
companion volume to his 'Statics,' and the two treatises together will carry a student as

far as he needs to go for all except special purposes A special feature of the book is

the avoidance of what the author rightly calls 'algebraical and trigonometrical puzzles
in disguise,' which are so often found among the examples for exercise. Here we find
questions of theoretical and practical interest, such as lead to a result worth the trouble
of solving, and not to a mere barren coincidence." — The Manchester Courier

Photo-Electriciiy. By Arthur Llewelyn Hughes, D.Sc, B.A., Assistant
Professor oj Physics in the Rice Institute, Houston, Texas.

Demy Svo. pp. viii+144. With 40 text-figures. Price 6s. net

Since the account of the photo-electric effect given by R. Ladenburg
in the yahrbuch fiir Radioaktivitcit for 1 909, no complete resume of the
subject has been written. During the past five years, however, consider-
able progress has been made, and it is therefore thought desirable to give
some account of the condition of the subject at the present time. Such an
account is attempted in this book, all forms of ionisation by light being
considered, whether in solids, liquids or gases. — Extract from the Preface

"This volume belongs to the CambriJge Physical Series, and to say that is enough to
commend it to all who need lucid and exact information on the subject which it dis-
cusses....A clear and admirable summary ot the work done up to date." — The Spectator

THE RESPIRATORY FUNCTION OF THE BLOOD

The Respiratory Function of the Blood. By "Joseph Barcroft, M.J., B.Sc,
F.R.S. Fellow of King's College^ Cambridge.

Royal Svo. pp. x+320. With a plate and 156 text-figures. Price 18;. net

" At one time," says the author, " most of my leisure was spent in
boats. In them I learned what little I know of research, not of technique
or of physiology, but of the qualities essential to those who would venture

Fig 133. — View of Matterhorn from the Capanna Margherita at sunset

beyond the visible horizon. The story 01 my physiological 'ventures'
will be found in the following pages."

The book is divided into three parts — I. The Chemistry of Haemoglobin^
II. The Passage of Oxygen to and from the Blood, III. The Dissociation Curve
considered as an '■^Indicator" of the ^^ Reaction" of the Blood. A specially
interesting portion is that which deals with the effect of exercise and of
altitude on the action of the blood.

"The investigation of the respiratory function of the blood is one of the most
difficult and yet one of the most interesting lines of physiological research, and
Dr Bancroft's volume is worthy of the subject. His work along these lines is already
well known, but the issue of it in book form is a welcome addition to the literature of

the subject The volume is more than a mere chronicle of experiments performed by

the author and his colleagues, though considered from this aspect alone the results are
indeed valuable. To the specialist in this branch it provides much matter of far-
reaching importance, and contains many valuable suggestions for further research." —
The Scotsman

10

SHAFTESBURY— ENGLAND AND GERMANY

Second Characters ; or, The Language of Forms, by the Right Honour-
able Anthony), Earl of Shaftesbury. Edited by Benjamin Rand,
Ph.D., Harvai'd University.
Demy 8vo. pp. xxviii + 182. With a frontispiece. Price ys. 6J. net

The publication of this book is the result of a discovery among the
Shaftesbury Papers of a MS volume containing the plan and fourth treatise
of a work intended as a complement to the famous "Characteristics."

" The book was to consist of four treatises. These were : I. ' A
Letter concerning Design ' ; IL 'A Notion of the Historical Draught or
Tablature of The Judgment of Hercules'; IIL 'An Appendix concerning
the Emblem of Cebes ' ; and IV. 'Plastics or the Original Progress and
Power of Designatory Art.'...' Plastics,' regarded by the author as the chief
treatise of the four, has never previously been published. The definite
grouping of these various treatises in the form of a single work, as intended
when written, is also here first made known."

Mr Edmund Gosse, writing in The Morning Post, says :

"Dr Rand has been a faithful servant to the memory of Shaftesbury. It was he
who, in 1900, discovered at the Record Office the treatise called 'Philosophical
Regimen,' and published his discovery. He now comes forward with a still more
important and interesting disclosure. ...Dr Rand, like a very clever architect, has found
pillars and capitals scattered by earthquake over a plain, and has so put them together
that they form, though still unhappily imperfect, the outlined structure of a temple.
...What is peculiarly interesting is the evidence given to us by the text of 'Plastics,'
here printed exactly as it was written, with regard to the author's style. This, in
Shaftesbury's revised works, published in his own lifetime, is polished to the extremity,
and, indeed, beyond the extremity, of elegance, worked upon till the surface is as smooth
and as frigid as marble. We are now able to observe how completely this last effect
was the result of labour after a false ideal, and how entirely different his style was
when it expressed his unsophisticated thought."

The Literary Relations of England and Germany in the Seventeenth
Century. By Gilbert JVaterhouse, M.A. Formerly Scholar oj St 'Johri s
College, Cambridge, First Tiarks University Gennan Scholar, English
Lecturer in the University of Leipzig.

Demy 8vo. pp. xx+190. Price -js. 6d. net
This work is inspired by Professor Herford's well-known volume on
the Literary Relations of England and Germany in the Sixteenth Century,
and investigates their nature during the century which followed. In the
earlier period the literary influence of Germany upon England was stronger
than that of England upon Germany, whereas in the eighteenth century
the reverse is the case. The object of the present work is to bridge the
gulf which lies between these two periods.

Contents : Introduction. Early Travellers. Earlier Lyrical Poetry. Sidney's
"Arcadia" in Germany. The Latin Novel. The Epigram. History in Literature.
English Philosophers in Germany. The Theologians. Later Travellers. The
Awakening of Germany and the Growth of English Influence. Later Lyrics. Later
Satire. Milton in Germany. Conclusion. Appendixes. Index.

II

NAVAL AND MILITARY SERIES

The first two volumes of tin's series, under the a:eneral editorship of
J. S. Corbett, LL.M., F.S.A., and H. J. Edwards, C.B., M.A., are now

ready.

Ocean Trade and Shipping. By Douglas Owen, Barrhter-at-Law, Lecturer
at the Royal Naval IFar College, and to the Army Clan the London
School of Economics and Political Science, formerly Secretary of the Alliance
Marine Assurance Co., Ltd.

Demy 8vo. pp. X + 27S. Witli 5 illustrations. Price loi. 6</. net

The author has endeavoured to supply in a single volume a summary
ot the "whole host of separate industries and undertakings" which come
under the head of Ocean Trade and Shipping.

The book is an explanatory work rather than a technical treatise.

The Baltic; view of "The Floor"

To army officers a knowledge of the machinery of shipping must be
interesting in view of the importance of sea transport as a military factor — -
especially in time of war.

Naval officers are brought into frequent contact with oversea trade, not
only in arranging for the supply of fleets and naval bases in time of peace,
but in the attack on, and defence of, commerce in time of war ; to his
summary, therefore, of the process of ocean trade in times of peace the
author has added some reflections on the situation created by a sudden
outbreak of war.

12

NAVAL AND MILITARY ESSAYS— NEW MANUALS

Naval and Military Essays. Being Papers read in the Naval and Military
Section at the International Congress of Historical Studies, 1 9 13.
Demy 8vo. pp. viii + 244. Price js. 6d. net

Contents: Historians and Naval History (by Sir J. K. Laughton,
Litt.D.). Staff Histories (by J. S. Corbett, LL.M., F.S.A.). Naval
History from the Naval Officer s Point of Vietv (by Capt. H. W. Richmond,
R.N.). Samuel Pepys as a Naval Official (by J. R. Tanner, Litt.D.).
Naval History and the Necessity of a Catalogue of Sources (by Lieut. Alfred
Dewar, R.N.). Appendix : Rough Guide to British Sources oj Naval History
in the Seventeenth Century. The Difficulties encountered in compiling Military
History (by Col. Sir Lonsdale Hale, late R.E.). The Value of the Study of
Military History as Training for Command in IVar (by Lieut. -Col. F. B.
Maurice). The Practical Application of Military History (by Lieut.-Col.
N. Malcolm, D.S.O.). Precis of the Plans of Napoleon for the Autumn
Campaign s/'iSlJ (by J. Holland Rose, Litt.D.). The Influence of Tactical
Ideas on Warfare (by L. S. Amery, M.A., M.P.). Field-Marshal Prince
Schwarzenherg : A Character Sketch (by Dr J. F. Novak). A Defence of
Military History (Synopsis of Paper read by Professor C. W. C. Oman,
M.A., F.B.A.). Foreign Regiments in the British Service, 1793-1815
(Synopsis of Paper read by C. T. Atkinson, M.A.). Index.

CAMBRIDGE MANUALS. Under the general editorship of P. Giles,
Litt.D., and A. C. Seivard, F.R.S. is. net each in cloth ; 2s. 6d. net in

leather. Six new volumes will shortly be ready : — ^

81. The San. By Prof R. A. Sampson, D.Sc, F.R.S. {Illustrated.)

Aims at giving the general reader a report upon the present position oi fact and
theory relating to the Sun.

82. Coal Mining. By T. C. Can trill, B.Sc {Illustrated.)

A sketch of the principles of coal mining, including an outline of the evolution of
the industry from its primitive beginnings ; an estimate ot its far-reaching effects on
domestic and mechanical affairs ; and references to the geological aspect where necessary.

83. The Making of Leather. By Prof H. R. Procter, M.Sc. {Illustrated.)
A brief history of the industry, with a description of modern conditions.

84. The Royal Navy. By John Leyland.

Gives a general view of the nature, character and development of the Navy.

85- Military History. By the Hon. J. IV. Fortescue.

A series of lectures — mainly on the British Army — originally given ^t Trinity
College.

86. Economics and Syndicalism. By Prof A. IV. Kirkaldy, M.A.

The chief need, in the author's view, is that all classes should be equally versed in
the laws which regulate the production, distribution, and consumption of wealth.

13

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The publications of The University of Chicago Press are sold in Great
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London in English Literature. By Percy H. Boynton. With 46 illustra-
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Unpopular Government in the United States. By Albert M. Kales.
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The evils of the present complex system of state and municipal government in the
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Animal Communities in Temperate America, as illustrated in the Chicago
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Masters of the Wilderness. By Charles B. Reed. i6mo. Price \s. net

Contains three essays entitled as follows : (i) The Masters of the Wilderness : A
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Materials for the Study) of Elementary) Economics. Edited by L. C.
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14

Books published since 29 'January 1914

THEOLOGY

Joshua.: The Hebrew and Greek Texts. By S. Holmes^ M.A., Lecturer
in Theology, "Jesus College, Oxford, and formerly Senior Kennicott Scholar.
Demy Svo. pp. viii + 8o. Price Js. net.

The result of Hollenberg's enquiry into the texts of the Book of Joshua in 1876
was in many passages favourable to the Lxx ; he strongly denies deliberate alteration
but on the whole seems to uphold the general superiority of the M.T.

Ten years later a far less favourable attitude was adopted by the great scholar
Dillmann ; he affirms that the value of LXX, in this book as well as in others, has been
much overestimated. Other scholars have given their support to this view, and
Holzinger (1901) explicitly affirms that the statement of Dillmann — that LXX does not
offer a more original text, but represents in many cases a deliberate endeavour to avoid
difficulties — still holds good.

The present thesis dissents from this position and offers some fresh reasons in favour
of the superiority of the LXX :

(1) The phenomenon of double and sometimes more frequent omission of the same
word or expression in LXX in a large number ot passages.

(2) The circumstance that in several cases where the two texts vary from one
another, each text is consistent with itself; thus suggesting the hypothesis of a deliberate
and systematic revision.

(3) The fact that the confused LXX passage, ch. v. 'w. 4 f , when turned back into
Hebrew requires only a slight emendation to give an intelligible text manifestly earlier
than M.T.

Evolution and the Need of Atonement. By Stewart A. McDowall, M.A.,
Trinity College, Cambridge, Assistant Master at Winchester College.
Crown "ivo. pp. Ar.v+184. Price i^s. bd. net.
A second edition, revised and enlarged. The author addresses his work to those
who are oppressed with the difficulties of their faith not as an apologetic, but as a re-
statement of certain fundamental doctrines of Christianity from that point of view
which the spirit of the age forces upon us. In the new edition a considerable section
has been added to the discussion of original sin, and the treatment of the Atonement
itself has been amplified by a new chapter linking the two portions of the book more
closely.

Nestorius and his place in the History of Christian Doctrine. By

Friedrich Loofi, D.D. See p. 4.

The Canticles of the Christian Church. By James Meams, M.A. See
p. 4.

The Poem of Job. ^ ranslated in the metre of the Original by E. G. King,
D.D. See p. 5.

The Fourfold Gospel. Section IL The Beginning. By Edwin A.
Abbott. Diatessarica, Part X, Section H. See p. 4.

15

THEOLOGY— LATIN

The Bible of To-Dayi. By A. Blakhton, M.A. See p. 5..

Parerga Copiica. Conscripsit Strphanus Gaselee, A.M., Collegii sanctae

Mariae Magdalenae apud Cantiihrigienses socius et hihliothccariui, et hoc
anno alter e Procuratorihus Academiae. IL De Abraha et Melchisedec.
in. Hymnvs de Sinvthio. Royal ^vo. pp. iv + 2^. Paper covers.
Price IS. bd. net.

The Offices of Baptism and Confirmation. By T. Thompson., M.A., of
Saint Ansfhns House, Cambridge. Small crown ^vo. pp. jr + 254.
Price 6s. net.
One of the Cambridge Huihlbooks of Liturgical Stitdy, under the general editorship of
Dr H. B. Svvete and Dr J. H. Srawley. Its aim is to trace the developments of the ser-
vices in general outline, and to indicate the relations of various rites to each other ; and
to help those who desire to study the services in the ancient liturgical books.

The Second Book of Kings. Edited by the Rev. G. H. Box, M.A.
Fcap. ?,vo. pp. xvi+l'-)i. Price is. bd. net.

Isaiah xl-lxi>i. Edited by the Rev. W. A. L. Elmslie, M.A., and the Rev.
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20

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Dr J. R. Clark Hall, author of a prose translation of Beoivulf, has
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22

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Complex Integration and Cauchy's Theorem, by Mr G. N. Watson,
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CONTENTS

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PAGE

R P. Gregory. Note on the Inheritance of Heterostylism in

Primula acaulis Jacq. . 303

R. P. Gregory. On Variegation in Primula sinensis. (With

Plates IX and X) 305

H. Drinkwater. a Second Brachydactylous Family. (With Plates

XI— XV, and 3 Text-Figures) 323

C. J. Bond. On the Primary and Secondary Sex Characters of
some Abnormal Begonia Flowers and on the Evolution of the
Monoecious Condition in Plants. (With Plates XVI and XVII) 341

E. RuGGLES Gates. On the Origin and Behaviour of Oenothera

ruhricalyx .......■■■• 353

Arthur Ernest Everest. Recent Chemical Investigations of the
Anthocyan Pigments and their bearing upon the Production of
these Pigments in Plants . 361

M. Wheldale. Our Present Knowledge of the Chemistry of the

Mendelian Factors for Flower-Colour 369

The Journal of Oenetics is a periodical for the publication of records of original
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