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Bulletin of the Museum of Comparative Zoology 
AT HARVARD COLLEGE, 
Vou: XX VI. No, at. 


SPERMATOGENESIS OF CALOPTENUS FEMUR-RUBRUM 
AND CICADA TIBICEN. 


Br E. V. Witcox. 


Wir Five Puates. 


CAMBRIDGE, MASS., U.S: A.: 
PRINTED, FOR THE MUSEUM. — 
Ae Mar, 1895. ae 


BULLETIN 


MUSEUM OF COMPARATIVE ZOOLOGY 


HARVARD COLLEGE, IN CAMBRIDGE. 


VOL. XXVII. 


CAMBRIDGE, MASS., U.S. A. 
1895-1896. 


UNIVERSITY PRESS: 


Joun Witson anp Son, CamBrineGg, U.S.A. 


CONTENTS. 


No. 1.—Spermatogenesis of Caloptenus femur-rubrum and Cicada tibicen. 
By E. V. Witcox. (65 Plates.) May, 1895 


No. 2.—On the Early Development of Limax. a C. A. Koroip. : Plates.) 
August, 1895 . 


No. 3.— Reports on the Dredging Operations off the West Coast of Central 
America to the Galapagos, to the West Coast of Mexico, and in the Gulf of 
California, in charge of ALEXANDER AGaAssiz, carried on by the U.S. Fish 
Commission Steamer “ Albatross,’ during 1891, Lieut. Z. L. Tanner, 
U. S. N., commanding. XVII. Birds from Cocos and Malpelo Islands, 
with notes on Petrels obtained at Sea. By C. H. Townsrenp. (2 colored 
lkinase)y sd lhuleey 2s Mg seo 6 cl as ac 


No. 4.— Reports on the Dredging Operations off the West Coast of Central 
America to the Galapagos, to the West Coast of Mexico, and in the Gulf 
of California, in charge of ALEXANDER AGassiz, carried on by the U.S. 
Fish Commission Steamer “ Albatross,” during 1891, Lieut. Z. L. Tanner, 
U. S. N., commanding. XVIII. Die Comatuliden. Von C. Harrraupe. 
(4 Plates.) August, 1895 ... . - 


No. 5.— Reports on the Dredging Operations off the West Coast of Central 
America to the Galapagos, to the West Coast of Mexico, and in the Gulf of 
California, in charge of ALEXANDER AGassiz, carried on by the U. S. Fish 
Commission Steamer “ Albatross,” during 1891, Lieut. Z. L. Tanner, 
U.S.N.,commanding. XIX. Die Ostracoden. Von G. W. Murier. (3 
Plates.) October, 1895 . sy Hh natch ‘ af BoM, © fue kc 

No. 6.—Studies in Morphogenesis. IV. A Preliminary Catalogue of the 
Processes concerned in Ontogeny. By C. H. Davenport. November, 1895 

No. 7.—The Early Embryology of Ciona intestinalis, Fleming (L.). By W. 
KE. Castuse. (18 Plates.) January,1896. .. . Pe re ott oi Reamer 


co 
co 


Bulletin of the Museum of Comparative Zodlogy 
AT HARVARD COLLEGE. 
Vou. XXVIII. No. 1. 


SPERMATOGENESIS OF CALOPTENUS FEMUR-RUBRUM 
AND CICADA TIBICEN. 


By E. V. Witcox. 


WitTuH Five PLATES, 


CAMBRIDGE, MASS., U.S. A.: 
PRINTED FOR THE MUSEUM. a 
May, 1895. 


No. 1. — Spermatogenesis of Caloptenus femur-rubrum and Cicada 
tibicen1 By E. V. Wicox. 


THE following observations were made on the testes of Cicada and 
Caloptenus. Only three male Cicadz were at my disposal, but of 
Caloptenus I examined more than twenty individuals. The Cicade 
were killed immediately after leaving the pupal case, and had been pre- 
served a number of years. The Calopteni were taken in August and 
September, 1893. 

The testes of Cicada were killed in Miiller’s fluid ; those of Caloptenus 
either in hot water, in hot corrosive sublimate, in cold corrosive subli- 
mate, or in chrom-osmic-acetic mixture. Some of the testicular folli- 
cles of Cicada were stained in Grenacher’s alcoholic borax carmine, 
others according to Bizzozero’s modification of Gram’s method. The 
follicles were stained im toto in safranin (50% alcohol) 24 hours, sec- 
tioned, stained 3 minutes in gentian-violet, washed 5 minutes in a solu- 
tion of potassic iodide, then treated alternately with alcohol and chromic 
acid (0.1%). But better results were obtained by double staining with 
safranin and victoria-green. Crystals of the latter were dissolved in 
absolute alcohol, or in clove oil. The sections were first stained in 
safranin (10-15 minutes), the excess of stain being quickly washed off 
in 90% alcohol, and then in’a very strong solution of victoria-green in 
absolute alcohol for 1 to 2 minutes. Staining and dehydrating were 
thus accomplished at the same time. Excess of green was washed out 
with absolute alcohol. Sections were cleared in clove oil. When a 
clove-oil solution of the green was used, the sections were dehydrated 
before staining in the green. The method with the absolute-alcohol 
solution gave the better results, and was more easily managed. 

The Caloptenus material was all stained on the slide. The methods 
used were either safranin and victoria-green, as just described, Henne- 
guy’s (91) potassic permanganate and safranin, or Heidenhain’s (92) 
iron-hematoxylin. The method with safranin and victoria-green gave 
good results. Cytoplasm and achromatic nuclear parts were stained 
green, the chromosomes, nucleolus, and centrosomes red. If the green 


1 Contributions from the Zodlogical Laboratory of the Museum of Comparative 
Zoology at Harvard College, under the direction of E. L. Mark, No. XLVII. 
VOL, XXVUI. — NO. 1. 


4. BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


be allowed to act too long, it will replace the safranin entirely. This 
(safranin and green) method was the only one by which the archoplasm 
was made distinct. The granular cytoplasm was stained green, and in 
the pale green clear areas of the archoplasm were to be seen the red 
centrosomes. In some stages the chromosomes were stained green, indi- 
cating that a chemical change takes place in the chromatic substance. 
But even in such cases the nucleolus was bright red. 

In using Henneguy’s method, the sections were put into permanganate 
of potash for 5 minutes, and then stained 3-20 minutes in Zwaarde- 
maker’s safranin. The mordant gives an iron-rust color to the sections, 
and the safranin must not be too much washed out, or the sharpness of 
outlines will be lost. It is best to wash out the mordant very thor- 
oughly before using the stain, for the potassic permanganate makes a 
precipitate with the safranin which renders the sections so muddy as to 
be nearly useless. By this method the chromosomes and nucleoli are 
stained bright red, the individual chromosomes being sharply outlined. 
In the metamorphosis of the spermatid, the six spheroidal chromatic 
elements are often easily distinguished, although closely massed to- 
gether. The chromatic crescent of the spermatid is very well defined ; 
but the small body in the neck of the spermatozoon, so conspicuous 
after treatment by Heidenhain’s method, is hardly to be seen when this 
method is used. Centrosomes were rarely stained ; achromatic fibres of 
ring stages were faintly stained; the nucleus often appeared as a clear 
lenticular space, in which were the red chromosomes. 

The best results were obtained by use of Heidenhain’s method. The 
“black” process proved more serviceable than the “blue.” The only 
mordant used was double sulphate of iron and ammonia, NH,Fe.(SO,),. 
A 2% aqueous solution was used as mordant, and a 4% aqueous 
solution as decolorizer. To produce the “blue” stain, the sections 
were placed in the mordant }—-1 hour, and after washing in water 
were stained in the hematoxylin (0.5% pure hematoxylin in H,O) 
1-2 hours ; finally, they were washed again in water. Sometimes it is 
necessary to decolorize a short time, say 20 minutes, in 4% NH4Fe.(SO,4)4. 
The “black” stain was obtained by leaving the sections in the mordant 
2 hours before washing in water, staining 10-12 hours in the hema- 
toxylin, and decolorizing 2-8 hours, finally washing as before. For 
either process the sections should be very thin. They must be firmly 
affixed to the slide; for the washing is best done by a stream of tap- 
water allowed to run over the slide. Three washings are necessary, 
each of which should be thorough: (1) after use of the mordant, 


WILCOX : SPERMATOGENESIS. 5 


(2) after staining, (3) after decolorizing. Simple immersion in water 
does not do as well. The mordant and stain will form a precipitate, 
just as in Henneguy’s method, and if the first washing be neglected, it 
is next to impossible to remove the precipitate by subsequent washings. 
One to five minutes in a stream of water is enough for each washing. 
The sections will become quite opaque immediately after immersion in 
the decolorizer, but in this the opacity is slowly removed. The decolori- 
zation is hastened by washing the sections in water at intervals during 
the process of decolorizing. This is necessary, also, in order to see how 
far the decolorizing has progressed. The process can thus be stopped 
at the desired stage. The proper decolorization is the most difficult 
part of this method. 

By the “blue” process, so far as my experience goes, the cytoplasm 
stains gray, the centrosomes do not stain at all, the spindle and linin 
fibres very faintly, the chromosomes dark blue. By the ‘ black” pro- 
cess the cytoplasm takes a dark-gray color, and both centrosomes and 
chromosomes are made black, while spindle fibres and linin fibres be- 
come very distinct. The nucleoli are colored nearly black by either 
process. 


CICADA TIBICEN. 


The testes of Cicada tibicen are paired, and each consists of a large 
number of ellipsoidal follicles, which are closely packed together. The 
follicles of each side of the body open into a vas deferens, which soon 
joins its fellow of the opposite side. Figure 14 (Plate I.) gives an idea 
of the spatial relationship to one another of different spermatogenetic 
stages. It represents a very nearly longitudinal section of a follicle of 
Cicada. At a are spermatogonia; at d, spermatids in various stages of 
metamorphosis. 

The Cicada material at my command did not show the division stages, 
but it gave a very reliable series of preparations on certain other stages. 

The spermatogonia lie at the blind end of the follicle. They occupy 
in my preparations only the single end-compartment (Fig. 14, a). Their 
size is less than that of the spermatocytes, and they are further distin- 
guished from them by the fact that they have only 12 chromatic rods, 
whereas the spermatocytes have each 24 spherical chromosomes. One 
or often two nucleoli are to be seen. 

The spermatocytes occupy usually two compartments next to that of 
the spermatogonia. The chromatic substance consists of about 24 


6 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


spheroidal bodies. In Figure 14 the compartment } contains spermato- 
cytes of the first order ; c, spermatocytes of the second order and sperma- 
tids just after the last division. (Compare Fig. 23 and Explanation of 
Figures.) The cells of 6 each contain one or two bodies which I con- 
sider nucleoli, since they react to the stains quite differently from the 
chromosomes. Figures 53-59 (Plate II.) represent cells quite commonly 
met with among the spermatocytes ; they are numbered in the order in 
which I think they succeed one another. In a single compartment may 
be found spermatocytes in several different conditions; the earliest 
seems to be that in which the nucleolus lies in the centre of the nucleus 
with the chromatic spherules arranged radially about it (Fig. 53). The 
nucleolus then moves to the periphery of the nucleus, and appears mean- 
time to have divided into two portions (Figs. 55, 56), one of which 
passes into the cytoplasm, while the other remains in the nucleus (Figs. 
58, 59); later, both parts appear outside the nucleus and on diametri- 
cally opposite sides of it. 

Hertwig (90) has noticed the aie ae of the nucleoli in the 
spermatocytes of Ascaris megalocephala just before the appearance of the 
centrosomes. Brauer (’93) figures the centrosomes as arising singly in 
each nucleus and dividing either inside (univalens) or outside (bivalens) 
the nucleus, according to the type. But Brauer saw nucleoli in the 
same nucleus with the centrosomes and differing from them in stain- 
ability. Born (94) maintains that the nucleoli have nothing to do with 
either reproduction or cell division. He says: “Die Nucleolen stehen 
in Beziehung zum individuellen Zellleben, nicht zur Fortpflanzung, denn 
beim Beginn der Mitose verschwinden sie um nach Beendigung der- 
selben —im Ruhezustand des Kerns — wieder aufzutreten.” 

Thus the nucleoli have been supposed to give rise to the centrosomes, 
to be modified chromatin, — a stage in the evolution of a chromosome, — 
to be excretory organs of the nucleus (Hicker, ’93), or to serve some 
unknown function in the economy of the cell (Born, 94). The nucleoli 
are found by Born to be very numerous and large in the germinative 
vesicle of the egg of Triton during the time when the chromatin is 
inconspicuous ; but they disappear entirely before the formation of the 
first polar globule. 

Since there is such disagreement about the origin, function, and 
fate of the nucleoli, it is probable that different structures have been 
called nucleoli by different authors. The several bodies in Cicada seen 
in and near the nucleus in Figures 50 and 53-61 (Plate II.) —in 
Figures 53, 54, as a single body, in Figures 55, 56, as two bodies, in 


WILCOX : SPERMATOGENESIS. 7 


Figures 57-59 as two bodies, one of which is outside the nucleus, and 
in Figures 50, 61, as two bodies, both outside the nucleus — seem to 
me to give evidence of being stages in the history of one and the same 
body. My reason for thinking that they are genetically connected is 
their similarity in size, structure, and reaction to stains. 

During the stages shown in Figures 49, 51, 52, there appears to be a 
chemical change in the constitution of the chromosomes. By the 
safranin and victoria-green method the chromosomes stain red, though 
not so deeply as the nucleoli. At later stages the chromosomes assume 
a green color, while the nucleoli continue to stain red. In still later 
stages (as Figs. 50, 60, 61) the chromosomes again take the red. 

The metamorphosis of the spermatid could be worked out in consider- 
able detail. The chromatin is first arranged around the periphery of 
the nucleus (Plate I. Figs. 24, 27-30). The individual chromosomes 
fuse into a thin shell of chromatin, surrounding, in part, the nuclear 
space. This chromatic shell does not extend over the whole periphery 
of the nucleus, and yet it is so extensive at the beginning of the meta- 
morphosis that in certain views of the nucleus it has the appearance of 
a complete sphere. 

Figures 15-18 and 24-45 show various stages in the spermatid meta- 
morphosis. Figures 62-77 (Plate II.) present a series of the changes 
which take place in the head of the spermatid. The stage in which the 
chromatin (Figs. 66-72) has the form of a crescent is very common, and 
therefore undoubtedly of considerable duration. 

The origin of the extranuclear body (Nebenkorper), which is stained 
dark green in Figures 20, 27-30, could not be determined. On the an- 
terior end of the nearly mature spermatozo6n (Fig. 1 ¢) is to be seen a 
highly refractive curved tip. Just behind it is a small darkly stained 
body. The body so conspicuous in the neck of the spermatid of Calop- 
tenus (Plate V. Figs. 196-200) was very rarely seen in the Cicada, 
probably because the methods used on Cicada would not stain it. 

Degenerating cells are very frequent in the testicular follicles of Cicada. 
So far as my work on Cicada and Caloptenus goes, amitotic division and 
degeneration affect only the spermatogonia, i. e. if the reproductive cell 
reaches the spermatocyte stage, it completes its course. The first sign 
by which I was able to recognize that a spermatogonium is becoming 
abnormal is due to a chemical change in the nucleus. The chromosomes 
stain more brightly than in normal cells. The cytoplasm becomes 
clearer and more homogeneous. Then the chromosomes become irreg- 
ular in shape, lose their individuality and fuse into a single mass, as in 


8 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


Plate I. Figs. 7, 19 (see Explanation of Figures), Plate V. Figs. 204, 
210, 211). This mass may be slightly vacuolated and may show nu- 
cleoli. Some stages of degeneration in the testes of Caloptenus are 
shown in Plate V. Figs. 202-220. The nucleus may divide amitotically 
once, twice, or three times (Figs. 206, 212, 214, 215). This may result 
in ragged granules or strands, or in regular chromatic spheroids (Figs. 
205, 207, 220). It is evident from Figures 202-220, since all are 
drawn to the same magnification, that some degenerating cells increase 
enormously in size. ‘These degenerating cells are very numerous in 
Cicada and Caloptenus, especially in the latter. 

In Cicada there are frequently seen spermatozoa and various stages 
in the metamorphosis of spermatids which are four or five times as large 
as the corresponding normal forms; they may be called giant spermato- 
zoa. Normal spermatids are represented in Plate I. Figs. 2, 3, 4, 9. 
Stages in the metamorphosis of giant spermatozoa, drawn to the same 
magnification, are shown in Figures 12, 13, 21, 22. Figures 78-103 
(Plate II.) represent giant spermatid cells found accompanying normal 
cells. Figure 104 shows normal spermatids, the magnification being 
the same as in Figures 78-103. The striking similarity between the 
corresponding stages of a normal spermatid and giant forms is very 
readily seen on comparing Figures 62-77 with Figures 78-86. The 
first series is much more highly magnified than the second, hence the 
apparent equality of size. 

Figures 202, 203 (Plate V.) represent the only examples found in 
Caloptenus which resemble the giant spermatozoa of Cicada. 

What is the meaning of giant spermatozoa? Frenzel (91, ’91*), Lowit 
(791), vom Rath (’90, 791, 793), Verson (91), Ziegler (’91), Ziegler und 
vom Rath (’91), Flemming (’89), Geberg (’91), and Meves (91), have 
discussed the question of amitotic division, Lochkerne, Ringkerne, and 
other degeneration conditions. I believe with vom Rath (793), that 
“Alle Zellen welche einmal amitotische Kernteilung erfahren haben, 
kénnen sich unter keiner Bedingung mehr mitotisch teilen, sie gehen 
vielmehr einem sicheren Untergang entgegen.” I believe that the 
giant spermatozoa are not functional, that they are excluded from the 
developmental series and really come to naught. But they arise in 
Cicada directly from spermatogonia without cell division, by a meta- 
morphosisis of the nucleus, which may or may not be accompanied by 
amitotic division of the nucleus. In such spermatogonia the nuclei may 
divide amitotically two or more times (Fig. 8). Then, in the majority 
of cases, the chromatin breaks up into numerous fragments, which are 


WILCOX : SPERMATOGENESIS. 9 


scattered about the cell irregularly after the nuclear membrane degen- 
erates. Or the fragments may fuse into one mass, which subsequently 
breaks up. But quite often in Cicada the metamorphoses of these 
nuclei are rather regular, presenting stages very similar to those which 
the normal spermatid undergoes. Iam not aware that any one hitherto 
has suggested that the giant spermatozoa arise directly from sperma- 
togonia, and a prior: it seems, I admit, quite improbable ; yet my Cicada 
preparations point very strongly to this conclusion. 


CALOPTENUS FEMUR-RUBRUM. 


The testes of Caloptenus consist of tubular follicles, which lie closely 
packed together, parallel to one another. They are of nearly the same size 
throughout their length, being slightly larger near the blind end of the 
tubule, and tapering thence into the collecting duct, which opens into 
the vas deferens. In my Caloptenus material, taken in August and 
September, the spermatogonia were confined to a single compartment at 
the blind end of the tubule. After the spermatogonia the other stages 
follow in regular succession, a considerable part of the follicle being 
occupied by the prophases of the first division of the spermatocytes. 
Then follow regions in which the two successive cell divisions are taking 
place, then the spermatid metamorphosis, and finally the nearly mature 
spermatozoa, which with the degenerating cells entirely fill the lumen 
of the tubule. Figure 108 (Plate III.) represents a longitudinal section 
of a follicle, in which spermatogonia are shown at a, prophases of the 
first spermatocyte division at 6, the first division at c, spermatids at d, 
immature spermatozoa at e, and degenerating cells at f. The stages of 
spermatogonia preparatory to division are seen in Plate III. Figs. 105— 
107, and Plate IV. Figs. 164-168. Spermatogonium divisions are 
shown in Plate III. Figs. 119-121, 124, 131, 138, and Plate IV. Figs. 
169-171, and a tripolar division at Plate IV. Fig. 189. I could not 
determine how many divisions the spermatogonia undergo. The chro- 
mosomes in the prophases are twelve in number, twenty-four at the 
equator of the spindle, during metakinesis. The individual chromosomes 
are rod-shaped or often elongate spindle-shaped. In metakinesis they 
show ordinarily the well known V-shaped figures, and are connected with 
each other in pairs by means of linin fibres. The centrosomes are usually 
apparent (Plate III. Figs. 105, 132). Figure 105 shows the centrosome 
surrounded by a clear protoplasmic area. In most cases a nucleolus is to 
be seen during the prophases. In Figure 106 there is in the nucleus a 
body (nucleolus?) which seems to have recently divided. 


10 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


Before describing the history of the spermatocytes, I will call atten- 
tion briefly to the figures which illustrate their various conditions. The 
earliest stage of spermatocytes that I have found is shown in Plate III. 
Figs. 110, 111. In Figure 111 the cell is not complete, part of the 
chromatin having been cut away in the previous section. Figure 221 
(Plate V.) is of about the same age. Figures 114, 116 (Plate III.), and 
184-186 (Plate IV.) give an idea of a slightly older stage. Figures 
187, 188 (Plate IV.), and 222 (Plate V.) are still older. In these the 
chromatic thread is already broken up into segments. Figures 228, 
229, 242, 243 (Plate V.), and 175, 176, 178 (Plate IV.), show various 
stages in the ring and “ Vierergruppen” formation. During the first . 
spermatocyte division the chromatic Vierergruppen are arranged at the 
equator of the spindle, essentially as shown in Figure 175 (Plate IV.). 
Figures 237-241 (Plate V.) illustrate the first division of the sperma- 
tocytes. 


The history of the spermatocytes of the first generation is as follows. 
(Compare Diagrams 3 to 6 and 7 to 10.) In the earliest prophase that 
I have seen (Plate III. Figs. 110, 111) the chromatic substance con- 
sists of numerous small granules, already arranged along a thread of 
substance, which itself stains somewhat (Diagram 3). The chromatic 
granules gradually become collected at twenty-four points on the thread 
(Diagram 4). The thread then breaks transversely into twelve seg- 
ments (Diagram 5). Each of these segments has the form of a dumb- 
bell, i. e. consists of two terminal chromosomes connected by a thread, 
composed of numerous linin fibres (Plate IV. Fig. 187, Plate V. 
Figs. 242, 243). 


WILCOX: SPERMATOGENESIS. 11 


The dumb-bell figures become associated in pairs (Plate V. Figs. 229, 
242, 243). Each of the six groups (Diagram 6) thus formed has the 
value of four chromosomes, each dumb-bell being equal to two chromo- 
somes (Plate IV. Fig. 178, Plate V. Figs. 242, 243). These quadri- 
valent groups may be formed by the approximation of the pairs of dumb- 
bells in one or the other of two ways. LEHither they become arranged 
quite irregularly (Plate III. Fig. 116, Plate V. Fig. 229), or the pairs 
may at first lie across each other at right angles, and later come to be 
parallel (Plate V. Figs. 229, 242, 243). A comparison of the draw- 
ings last mentioned will show how by the fusion of the ends of the two 
parallel dumb-bells a ring results, such as is shown in Plate IV. Figs. 
178, Lio, L3t: 

By a slight variation in the time at which the massing of the chroma- 
’ tin granules takes place the process up to this point may pursue a course 
apparently quite different from that described. The chromatin granules 
of the original chromatin thread do not become massed into definite 
chromosomes as early as in the method just outlined. Consequently 
the transverse divisions result in the formation of twelve segments 
(Diagram 8) with very irregularly serrated edges. These segmenta asso- 
ciate themselves (Diagram 9) in pairs (Plate V. Figs. 201, 225, 227). 
They are either so closely applied to each other as to appear like single 
rods, or else show two rows of granules (Fig. 227), and thus give the 
same appearance that would have resulted from a longitudinal splitting 
of a single segment. The component halves of these six segments sep- 
arate from each other except at their ends, and thus form rings, as in 
Plate IV. Fig. 174. The granules scattered along these rings then 
collect into four chromosomes (Diagram 10). The result is, therefore, 
the same as by the process first mentioned. 

This account of the formation of rings varies somewhat from those 
of vom Rath (93) and Hiicker (93), and is entirely different from 
Brauer’s (93) account. These differences, as well as the points of agree- 
ment, will be discussed under the literature of the subject. 

The position of the chromatic rings at the equator of the spindle is 
shown in Plate V. Figs. 192-195 and 237-241. The rings are always 
complete at this stage, and the first step in the metakinesis of the sper- 
matocytes consists in a separation of the rings into half-rings. With 
the iron-hematoxylin method the majority of the spindles present the 
appearance of Figures 194,195. The planes of the rings all pass through 
the axis of the spindle. Hence it is impossible to see that the chromo- 
somes are arranged in rings, except when the rings are turned broadside 


1h BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


toward the observer, as sometimes happens (Figs. 237-239). Figure 
192 shows four rings seen from the edge and two from the side. The 
four chromosomes of a group may be arranged in a square rather than 
in a circle (Plate IV. Figs. 175, 176). 

The division of the rings may present different appearances according 
to the position of the chromosomes with reference to the poles of the 
spindle. The group may be a square, 
with one side turned toward each pole of 
the spindle (Diagram 2), or it may be 
more diamond-shaped (Diagram 1) with 
an angle directed toward each pole. In 
either case division takes place as indi- 
cated by the dotted line, and the chromo- 
somes a and 6 go to one pole, c and d to - 
the other. The final result, therefore, is 
the same as before. But in the first case 
the chromosomes a and 8, still held to- 
gether by linin threads, move toward the pole maintaining unchanged 
their relative positions, i. e. the rod with a chromosome at either 
end remains at right angles to the polar axis of the spindle, and is 
therefore in proper position for the second division, which follows di- 
rectly upon the first, and is at right angles with it, a going to one sper- 
matid, 6 to the other. By the second mode the pair a, 6 starts for the 
pole, either in a very oblique position or nearly parallel to the polar 
axis, and with a in advance. It therefore must turn 45° or more so as 
to be in the proper position for the second division. 

The later stages of the first spermatocyte division are shown in 
Plate III. Figs. 112, 113, 117, 118, 122, and 123, which are drawn from 
preparations stained with safranin and victoria-green, or by Henneguy’s 
method. These methods do not bring out the individual chromosomes 
at this stage. Figures 118 and 122 show the interzonal filaments 
still bridging over the space between the already separated cells. Some 
cells at this stage (Figs. 113, 117) have a peculiar appearance, as if 
the division were amitotic. But the interzonal filaments between the 
two chromatic masses show it to be a mitotic division. 

The second division of the spermatocytes is shown in Plate III. 
Fig. 128, and Plate V. Figs. 190, 191, 231. This division is accom- 
panied by the formation of a typical spindle and centrosomes ; it effects 
a separation of the constituent chromosomes of each chromatic dumb- 
bell, and therefore results in giving each spermatid six univalent spher- 
ical chromosomes, such as are shown in Plate III. Figs. 125, 126. 


WILCOX: SPERMATOGENESIS. 13s 


The number relationships of the chromosomes in the spermatogenesis 
of Caloptenus may be thus tabulated : — 


Spermatogonia . . . . . . . . 12 univalent chromosomes. 
Spermatocytes, Ist order . . . . . 6 quadrivalent chromosomes. 
Spermatocytes, 2d order . . . . . 6 bivalent chromosomes. 
Spermatids ......- . . . 6univalent chromosomes. 


Expressed in individual chromosomes : — 


Spermatogonia. . .. . 12 Spermatocytes, 2d order . 12 
Spermatocytes, lst order . 24 permatids rs /ys et} .s 2 a ~-G 


Spermatids immediately after the second spermatocyte division are 
shown in Plate III. Figs. 125, 129, and Plate V. Fig. 232. There is at 
first no nuclear vacuole surrounding the six small spherical chromo- 
somes, which are closely packed together, and immediately surrounded 
by the granular cytoplasm (Plate III. Figs. 125, 126,129). The in- 
terzonal filaments are still to be seen, forming a striated body, probably 
the beginning of the “‘ Nebenkern,” as suggested by Platner (’86). 

Some of the spermatids stained by Henneguy’s method, and nearly 
all of those stained by Heidenhain’s method, show a spherical body near 
the chromatic mass (Plate V. Figs. 232-235), and this body becomes 
included in the nuclear vesicle when a membrane is formed (Plate IV. 
Figs. 140, 141, Plate V. Figs. 232, 236). I regard this body as the 
centrosome which is left in each spermatid after the last spermatocyte 
division, and I also believe it to be identical with the very conspicuous 
body which forms the neck of the spermatozoén (Plate V. Figs. 196-200). 
The chromatic substance fuses into a smoothly contoured mass, which 
soon assumes the crescent shape so common in insect spermatogen- 
esis. The neck-body lies within the nuclear membrane opposite the 
concavity of the chromatic crescent (Figs. 198-200). The chromatin 
undergoes chemical and physical changes during the metamorphosis of 
the spermatid, but the neck-body remains practically the same in size, 
and does not alter its affinity for stains. It becomes the neck of the 
spermatozo6n (Plate IV. Figs. 139-158, Plate V. Figs. 196-200). The 
chromatic crescent is at first less dense, and stains less deeply ; then it 
becomes concentrated, and stains nearly black by Heidenhain’s method. 
These changes in density are not well shown in the figures. At the 
same time it becomes elongated, one end applying itself to the neck- 
body, the other becoming the tip of the spermatozo6n head. 

The nuclear vacuolation, much reduced, persists for some time near 
the neck-body (Fig. 196), then disappears entirely, and the further 


14 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


metamorphosis of the head consists largely in an elongation. By Hen- 
neguy’s method the neck-body is only rarely stained. This explains its 
absence in Figures 142-154. By Heidenhain’s method it becomes in 
all spermatids a very conspicuous black spherical body (Plate V. 
Figs. 196-200). 


I pass now to a consideration of the results reached by other students 
of spermatogenesis in insects. 

Sabatier (790) has discussed in a short article the spermatogenesis of 
the Locustide. The conclusions to which he comes are rather startling. 
In regard to the metamorphosis of the spermatid, he says: “ Pres du 
noyau mais non a son contact immédiat apparait dans Je protoplasme une 
vésicule sphérique . . . la vésicule protoplasmique.” Sabatier main- 
tains that there is an almost total degeneration of the nucleus, but ad- 
mits that it gives rise to the “ Kopfkappe”: “Cette derniere dérive 
donc du noyau et fournit un exemple remarquable de la dégénérescence 
ou altération du noyau de la cellule spermatique.” It is quite remark- 
able that the nucleus is able to form only the Kopfkappe when, presto, 
“cette vesicule devenue fusiforme et vivement colorable constitue ce que 
Yon considére comme la téte du spermatozoide.” 

I have never seen any such nuclear degeneration, nor any extra- 
nuclear vesicle of such paramount importance. ‘The head of the Calop- 
tenus spermatozodn arises from the six chromosomes inherited by each 
spermatid. 

Blochmann (’87) describes the formation of the polar bodies in Blatta 
germanica. 

The work of vom Rath (’91* and ’92) on the spermatogenesis of 
Gryllotalpa must receive special notice. My account of the spermato- 
genesis of Caloptenus confirms a large part of vom Rath’s' results, but 
differs from his in several points, and suggests another interpretation 
of the last two divisions. What these differences are will soon be 
apparent. 

Vom Rath (’93) has already called attention to the many groups 
of animals in whose spermatogenesis or oOgenesis ring formation and 
Vierergruppen have been observed by different authors; but with one 
exception (Flemming, ’87) he mentions those authors only who have 
noticed and remarked about these chromatic figures. It may seem ven- 
turesome, but I wish to suggest the same interpretation for the figures 
of various authors who either had no definite idea of Vierergruppen, or 
considered the conditions exhibiting them abnormal (Flemming, ’87), 


WILCOX: SPERMATOGENESIS. 13 


and were thus unprejudiced in favor of rings or Vierergruppen in mak- 
ing their drawings. The older works will be mentioned first, and the 
important works of Boveri (’90), Brauer (93, ’94), Hacker (93), Hen- 
king (90, 91, 92), and vom Rath (’91*, ’92, ’93), will be considered 
later. 

Flemming (’87, pp. 444, 445) saw Vierergruppen in the Salamander. 
Figures 46-50 of his paper show chromosomes arranged in groups of 
four, the groups being scattered quite irregularly over the spindle, 
much as vom Rath figures them in his latest paper (’93). Flem- 
ming considered this arrangement as abnormal: “Sie [the group- 
ing into fours} kann wohl in der That als eine Anomalie bezeichnet 
werden, obwohl ich noch nichts daritber weiss ob aus den Folgestadien 
etwas normales werden kann oder nicht, . . . es finden sich also Grup- 
pen von je vier Kiigelchen von denen je zwei aneinanderhangen. Diese 
liegen anscheinend ganz regellos tiber die ganze Spindel hingestreut, 
nur offenbar mit der Tendenz sich nach den Polen anzuhiufen.” Vom 
Rath calls attention to Flemming’s explanation of these figures, and 
holds, quite rightly, that the groups are moving, not as Flemming 
imagined, toward the poles, but toward the equator, there to be separated 
into bivalent dumb-bells. Flemming believes he finds a tendency to 
irregularity in those spindles which bear four-grouped chromosomes, and 
considers such irregular spindles as so many stages in the degeneration 
of a bipolar spindle into a tripolar one. If with Flemming it is denied 
that the groups of four occur in the regular course of development, it 
must be concluded that these are degeneration stages. 

Platner (’86) has figured in Helix pomatia several stages of rings and 
their division without so interpreting them. Figure 4 of his article 
“Ueber die Entstehung des Nebenkerns,” etc., shows very clearly the 
ring condition previous to division. In his Figure 5 are groups of four 
chromosomes. Figure 12 shows rings on the equator of a spindle, and 
Figures 15-17 are metakinetic and dyaster stages, in which the spherical 
chromosomes are coupled into dumb-bell figures and some of the dumb- 
bells have rotated 90° and are ready for the second division, just as I 
have seen them in Caloptenus. 

I would call attention also to the following cases drawn from the 
literature of the subject : La Valette St. George (’85, Figs. 16, 17, ’86, 
Figs. 11, 21, 22), Zacharias (’87, Taf. VIII. and IX.), Kultschitzky (88, 
Fig. 3, and ’88*, Figs. 16, 17, 22), Carnoy (’85, ’86, and 86%), Guignard 
(91), Baranetzky (’90, Figs. 23, 26, 40), Hermann (’89, Fig. 31), 
Lukzanow (89, Figs. 21, 23), Henking (’92, Figs. 101, 153, 190, 216, 


16 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


217, 229, 413, 418), Stuhlmann (86, Figs. 228, 233), and Moore 
(93, Fig. 1). 

The terminology which I have used is that of La Valette St. George, 
as adapted by Boveri: — 


Spermatogonium = Hertwig’s Ursamenzelle. 
Spermatocyte, lst order = Samenmutterzelle. 
Spermatocyte, 2d order = a Samentochterzelle. 
Spermatid = es Samenenkelzelle. 
Spermatozoon. 


G. W. Field (’93) uses a terminology which seems to admit one less 
spermatocyte stage than is recognized by authors generally: “ We find 
that the largest cells, the spermatogones (using the nomenclature pro- 
posed by La Valette St. George and now very generally adopted), 
divide by mitosis and form two spermatocytes. Next each spermato- 
cyte divides, also by mitosis, forming two spermatids. Each spermatid 
then changes directly into the spermatozo6n, without further division. 
Thus each spermatogone gives rise to four spermatids.” Field uses this 
apparently as a general scheme of spermatogenesis. “ Spermatogones” 
are, I suppose, spermatogonia. But they are not “the largest cells” in 
Boveri’s scheme, nor do I find that La Valette St. George or any other 
author has applied the term to these large cells, which Boveri desig- 
nates as spermatocytes of the first order. The spermatogonia after they 
have ceased dividing as spermatogonia become by «' process of growth 
spermatocytes (Boveri’s spermatocytes, Ist order). Field’s “sperma- 
togones” therefore probably correspond to Boveri’s spermatocytes of 
the Ist order, his “spermatocytes” to Boveri’s spermatocytes of the 
2d order, and the spermatogonia of Boveri are unmentioned. Field has 
therefore extended the use of the term spermatogonia to cover the whole 
period of that cell generation which Boveri calls at its beginning the last 
generation of spermatogonia, and during the rest of its existence sper- 
matocytes of the first order; consequently he designates as a spermato- 
gonium division one that Boveri calls a spermatocyte division. It is 
difficult to see why the fact that “each spermatocyte divides also by 
mitosis”? need be so strongly emphasized. It would be much more 
strange if the spermatocytes divided amitotically (compare vom Rath 
91 and 793, and Ziegler 791). 

Henking (’91) in his paper on Pyrrhocoris has considered the origin 
and fate of the chromatic rings. His Figures 13-20 show stages in the 
formation of the rings. Henking differs from most other authors in 
denying that there is any doubling of the chromosomes between the last 


WILCOX : SPERMATOGENESIS. ay 


division of the spermatogonia and the first division of the spermatocytes. 
He maintains that the first division of the spermatocytes is a reduction 
division and the second an equation division. His number relationships 
for the chromosomes are hence the following : — 


Spermatogonia . . iste pits dw 4 
Spermatocytes, 1st ee 12 Faden’ rungs « . » 2 
SPSTINALOGY Leds SMe OLMER!: yi. cs:t hive, ven «wy ae 12 
DN CUMIAN CHIR oad crab 8 e Mish ps! pk Wy, ey, LD 


The only reference by Henking to rings of the value of four chromo- 
somes is in this sentence: ‘‘ Ich mache besonders auf die mit vier Ver- 
dickungen versehenen Ringe 1 und 2 in Fig. 20 aufmerksam.” The 
two rings to which Henking refers contain each four nearly spherical 
chromosomes, and these, I believe, are the only instances in which 
Henking recognized the real value of chromatic rings. Each ring con- 
tains four chromatic elements, each half-ring two elements, and since 
these two elements are separated from each other at the second sper- 
matocyte division, this, contrary to his conclusion, is just as truly a 
reduction division as is the first. But Henking objects to this in- 
terpretation: “ Es findet hier also keine Reduction statt, sondern eine 
gewohnliche Aequationstheilung, welche jedoch hier schon von fernher 
vorbereitet war.” But if each ring has the value of four, not simply 
two, chromosomes, the same argument could be applied to the first as 
well as the second spermatocyte division, as Brauer (’93) has already 
done. The soundness of these objections will be considered in connec- 
tion with Brauer’s paper. 

Hacker (’92* and ’93) has seen ring formation and Vierergruppen 
in the odgenesis of several marine Copepods. In the genera Eucheta, 
Calanus, Cyclops, Diaptomus, Canthocamptus, and Heterocope, he main- 
tains .hat “zwischen die letzte Theilung der Ureizellen und die erste 
Theilung der Reifungsphase ist kein feinfadiges Ruhestadium des 
Kernes (Keimblaschenstadium) eingeschaltet.” In the eggs of some 
females this resting stage is passed over, in others not. In those females 
in which the resting stage in odgenesis is twice omitted, i. e. both before 
and after the formation of the first polar globule, Hiicker (92) sug- 
gests, as a motive for the omission of the first resting stage, that in this 
way “im Mikrokosmus des regenerativen Lebens eine weitgehende 
Anpassungsfihigkeit zur Geltung gelangt.” This omission, then, is a 
biological adaptation. The maturation of the egg is thus brought about 
sooner. This explanation is mentioned, because it has a direct bearing 
upon any interpretation of the rings, as will soon be seen. Hicker 

VOL. XXVII. — NO. 1. 2 


a 


18 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


(93, pp. 462, 463) describes the formation of the Vierergruppen as fol- 
lows: “Die eigentliche chromatische Substanz konzentriert sich naém- 
lich zuniichst auf bestimmte Stellen der Doppelfadenschlinge, und an 
jeder dieser Stellen tritt mehr und mehr eine scharfe Knickung hervor 
(Fig. 15 a, 16 a), durch welche diese Doppelfadenpartieen je in zwei 
gleiche Schenkel getheilt werden. ... In einem weiteren Stadium 
findet eine tropfenformige Verdickung der 4 Enden der Doppelschenkel 
statt (Fig. 16 6 und c), in den Ecken der Doppelwinkel kommt es dann 
zur Zerlegung derselben (Fig. 16 d), die vier Schenkel verkiirzen sich 
noch mehr und das Resultat dieser Verinderungen sind demnach Biin- 
del von je vier kurzen dicken Stibchen, welche in der oben angegebenen 
Weise durch feine Doppelfiiden mit den Nachbarbiindeln verbunden 
sind. Es sind die charakteristische Vierergruppen welche immer wieder 
und wieder vor der ersten Theilung der Reifungsphase auftreten.” 
Hiicker’s theoretical. explanation of the Vierergruppen may be learned 
from the following: ‘“ Wenn wir unter einem Paar von Schwester- 
elementen [‘identischen Idanten,’ Weismann ’91] solche Elemente 
verstehen welche durch Lingsspaltung eines Mutterelementes entstanden 
sind, so besteht also jede Vierergruppe aus zwei Paaren von Schwester- 
elementen welche im zusammenhingenden Doppelfaden ursprunglich hin- 
tereinander gelegen sind.” Hicker (’92) had previously, in considering 
the longitudinal splitting of the chromatic thread in the prophases of 
the first division, treated this as a process by itself, and so had regarded 
both polar-body divisions as reduction divisions. But later he came to 
view this splitting as a precocious preparation for the formation of the 
first polar body, or rather as a process pushed back in time by the sub- 
sequent introduction of a germinative-vesicle condition. He now (93) 
sees in the first division a modified equation division : “‘ Um zu bewei- 
sen dass die erste Theilung eine modificierte Aequationstheilung ist, 
miissten wir zeigen dass ihr eine einmalige Lingsspaltung vorangeht 
durch welche die Normalzahl der Elemente verdoppelt wird, und dass 
dann bei der Theilung die so erzeugten Schwesterelemente auseinander- 
treten.” 

Hiicker’s conclusion with regard to the Vierergruppen is, in his 
own words: “ Heissen die im Chromatinfaden hintereinanderfolgenden 


[danten a, b,c . . ., so wiirde der lingsgespaltene Chromatinfaden sich 
nach Weismann durch is : F ay \ darstellen lassen, und die Formel 


fiir eine Vierergruppe ist : ae it . dJede Vierergruppe besteht also im 


WILCOX : SPERMATOGENESIS. 19 


Sinne Weismann’s aus zwei Paaren von Schwesterelementen und nicht, wie 
dies nach Boveri’s und Brauer’s Angaben der Fall sein wiirde, aus vier 


Enkelelementen ({ ales \ ).” 


aa 

The separation of the sister elements, which according to Hicker 
occurs in the first division, constitutes an equation division, and “ In der 
zweiten Richtungstheilung erfolgt dann die definitive Trennung der 
nichtidentischen Idantenpaare.” ‘Therefore, the second division alone 
is a reduction division. 

Vom Rath (91°, ’92, ’93) has in two important papers discussed the 
formation of rings and the meaning of the Vierergruppen in connec- 
tion with the question of reduction. Hacker and vom Rath agree in 
all essential points as to the origin of the rings, as can be seen from the 
following quotation from vom Rath’s last paper: “‘ In allen von mir un- 
tersuchten Fallen der Spermatogenese und Ovogenese entstehen die Vier- 
ergruppen vor der Reifungsperiode in gleicher Weise dadurch, dass im 
Kniauelstadium zwei hintereinander gelegene Segmente mit einander ver- 
bunden bleiben und mit den durch die Langsspaltung des Chromatinfa- 
dens entstandenen ebenfalls verbundenen zwei Schwestersegmenten eine 
bald innigere (Ringbildung) bald losere (keine Ringbildung) Zusammen- 
gehorigkeit bewahren. Aus jedem dieser vier Segmente entstehen dann 
durch Kontraction vier Stiibchen — oder Kugelchromosomen. Es scheint 
mir daher das Natiirlichste zu sein, jede Vierergruppe als aus vier 
Einzelchromosomen bestehend anzusehen.” Hiacker’s account, as pre- 
viously quoted, is strikingly similar to this. Vom Rath evidently meant 
to say that out of each of the four segments arises by contraction one 
spherical chromosome, not four. If four chromosomes arose from each 
of the four segments, we should have groups of sixteen. 

Vom Rath inclines to the belief that both maturation divisions are 
reductions : “ Wieder andere, nimlich Weismann, Hacker und ich, lassen 
die Reduction durch beide Theilungen erfolgen.” He adopts Hacker’s 


formula ( ; a A ), quoted above, for the Vierergruppen. The scheme 


of numbers of chromosomes, exactly the same as that of Hicker, is as 
follows :— 


For Gryllotalpa. For Salamandra. 
Spermatogonia, 12. ‘ 24. 
Spermatoc. Ist order, 6 rings, 24 chromosomes. 12 rings = 48 chromosomes. 
Spermatoc. 2d order, 6 half-rings, 12 “ 12 half-rings—=24 “* 


Spermatid, 6 chromosomes. 12 chromosomes. 


20 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


The number of chromosomes in the spermatocytes of the first order is 
thus double the normal number, and this is reduced to one half the 
normal number by two reduction divisions (vom Rath, ’92 and ’93). 

Brauer (’93) in studying the spermatogenesis of Ascaris has come to 
some quite definitely stated conclusions in regard to the meaning of 
the reduction of the chromatin, which can be made clear by a few 
quotations : — 

“Es ist zwischen beiden [spermatogonia and spermatocytes] ein 
echtes Ruhestadium des Kerns vorhanden, von einem Fortbestehen der 
Chromosome der Spermatogonien kann keine Rede sein.” 

“Es bedarf kaum einer niheren Auseinandersetzung, dass diese 
Ansicht sich mit der Individualititshypothese der Chromosome... . 
nicht vertriigt. Die Chromosome sind ftir mich keine selbstindigen In- 
dividuen, sondern sie sind nur die Verbinde fiir die zahllosen kleinen 
Chromatinkorner, welchen allein der Werth eines Individuums zukommt.” 

“Tst meine Ansicht tiber die Bedeutung der Chromosome richtig, so er- 
folgt bei diesen Theilungen keine Reduction der Zahl der Chromatinkor- 
ner, sondern nur eine solche ihrer Masse. Eine Reductionstheilung im 
Sinne Weismann’s findet mithin nicht statt. Eine solche diirfte meiner 
Ansicht nach iiberhaupt unméglich sein, wo wenigstens die Theilung auf 
karyokinetischem Wege erfolgt.” A like conclusion is reached for the 
centrosomes : “So miissen dieselben bei der Befruchtung in irgend einer 
Weise eine Reduction ihrer Masse erleiden, da sonst eine stete von Gen- 
eration zu Generation fortschreitende Vergrésserung eintreten miisste.” 

Speaking of the longitudinal splitting of the chromatin granules, he 
says: ‘Da diese Theilung aber das Wesen der ganzen Karyokinese 
ausmacht, so halte ich alle diejenigen Erscheinungen welche ihr folgen, 
wie das Ansammeln der Korner auf wenigen Faden, ihre Vereinigung zu 
grésseren Kérnern, der Zerfall eines Fadens in Segmente und schliess- 
lich in Chromosome, fiir weniger bedeutend.” Briefly, Brauer does not 
believe in any reduction division in the Weismannian sense of the term, | 
but only in a reduction of the mass of the chromatin. 

Thus there have been proposed by different authors four different 
solutions of the question of reduction. Henking holds that the first 
maturation division is a reduction, the second an equation division ; 
Hertwig considers the first an equation and the second a reduction 
division; Brauer maintains that there is no “reduction” in either 
division (except in mass), whereas Weismann, Hicker, and vom Rath 
maintain two reduction divisions. But Hacker (’93) now calls the first a 
“modified equation ” division, and only the second a reduction division. 


WILCOX : SPERMATOGENESIS. val 


I have used the word “reduction” without indicating the particular 
sense in which I use it. The definition of reduction proposed by Weis- 
mann (’92), and adopted by vom Rath, Hicker, and-others, is that which 
I prefer, and according to which I have used the term. This is: “ Unter 
Reductionstheilung verstehe ich eine jede Kerntheilung durch welche die 
Zahl der Ide welche im ruhenden Kern vorhanden war, fiir die Tochter- 
kerne auf die Hialfte herabgesetzt wird.” It is not necessary to adopt 
Weismann’s terms, ‘‘ids, idants,’”’ etc., in order to use his definition. If 
the developmental possibilities are only one half as great in the daughter 
nucleus as in the mother nucleus, there has been a reduction in Weis- 
mann’s sense. If a nucleus contains four elements which happen to be 
two pairs of identical elements, the formula would be 

n 


ale 
m 
Now, if the division takes place along the line # y, there is a reduction 
in Weismann’s sense ; but if the division be along the line m n, it is an 
equation division. Either division would reduce the chromatic mass, 
but only the first would reduce the number of diferent elements (ids) 
in the daughter as compared with the mother cell. 

Since the rings, or Vierergruppen, have already been found in the 
odgenesis and spermatogenesis of numerous species in different groups, 
this arrangement of the chromatin just before the maturation divisions 
is certainly very general, if not practically universal. In order, there- 
fore, to interpret properly these two divisions, and to come to any 
sound conclusions with regard to the reduction question, it is of funda- 
mental importance carefully to study the formation of the Vierergruppen. 
Hacker (’93) and vom Rath (’93) have already called attention to the 
fact that the double longitudinal splitting of the chromatic thread, main- 
tained by Boveri and Brauer, must bring about groups of four édentical 


elements. The formula for a Vierergruppe would then be tae 


There could not in this case be any Weismannian reduction in either 
division, for there is only one kind of 7d in all the four elements of the 
group. If the Vierergruppen always arose as Brauer describes the 
process, — i.e. by two longitudinal splittings of the chromatic granules, 
which alone, he believes, possess an individuality, — then the four com- 
ponents of each group would be identical, and there could be no reduc- 


2 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


tion division. Brauer therefore contends that a Weismannian reduction 
is impossible in karyokinetic division, and indeed never occurs. For 
Brauer the reduction question has consequently found its final solution : 
There is no reduction except merely one of mass. This would offer 
a very simple answer to the problem. But Henking (90, 791, ’92), 
Riickert (792, 93), Hiicker (91, 93), and vom Rath (91%, ’92, 93) have 
been able to see only ove longitudinal splitting. I have seen no evidence 
of any such splitting of the chromatic thread in Caloptenus. Since com- 
ing to that conclusion, I have read in Born’s paper on the egg of Triton 
(94) that he finds a doubling in the number of the chromatic elements 
during the germinative-vesicle condition, but this doubling does not 
result from a longitudinal splitting. The chromatic thread divides 
transversely into twice as many segments as there were chromosomes in 
the Ureizelle. Born’s statement that “eime Verdoppelung durch Quer- 
theilung stattfindet”’ is in essential agreement with my results. 

A remark by Wagner (92) —to the effect that twice as many 
chromosomes arise during the resting stage immediately before the first 
maturation division as were in the cells of the preceding generation, 
but that this does not imply any such definite longitudinal splitting 
as Brauer and other authors maintain — may also be interpreted as in 
harmony with my conclusions. 

Since the chromatin in the resting stage is very finely divided, — at 
least into finer particles than the ‘Chromatinkérner ” to which Brauer 
ascribes the dignity of individuality, —it seems to me just as arbitrary 
to consider these homogeneous “Chromatink6érner” units, which by di- 
vision must give rise to identical daughter grains, as to make the same 
supposition with regard to the chromosomes. 

But further, even if we grant for a moment that the Vierergruppen 
do arise by two longitudinal divisions of the chromatic granules, what 
evidence have we that each Vierergruppe consists of four identical 
elements? Brauer maintains that both these splittings take place very 
early in the resting stage. The granules are extremely small. Each 
chromatic quarter of the group increases considerably in size. This 
growth takes place while they are separated (held together only by 
linin threads). There is still the probability that chromatic substance 
is formed in the nucleus during the process, and becomes associated 
with the substance of the Vierergruppen. In order to insure the 
identity of the elements of a Vierergruppe, two longitudinal divisions 
must take place after all growth of the chromatin has ceased, and we 
must at the same time assume that the chromatic elements are homo- 


WILCOX: SPERMATOGENESIS. 23 


geneous, or, if they are not homogeneous, that there is an exact halving 
of the component particles of the elements of the Vierergruppe. But 
Brauer considers the four elements of a group identical because they all 
arise, by two divisions, from one. 

Again, if this whole process be only to secure a reduction of the mass 
of chromatin, the doubling of the chromatic elements, and the long, 
laborious process of mitosis would be unexplained and unjustified, as 
Weismann has pointed out; for a halving of the mass could be brought 
about by amitotic division. According to Weismann, the formula for 


Brauer’s Vierergruppe would be (Hicker, ’93) 1 ie We start 


with one element, @; this undergoes two longitudinal splittings, and 
then two separations by the two maturation divisions, and we then have 
just what we started with. The series formulated would be 


a 
a 


a 
a aa a 


a 
a 


But Henking, Weismann, Hicker, Riickert, vom Rath, and others 
allow only one longitudinal splitting, and their formula for the Vierer- 
gruppen, as stated by Hicker (93), and accepted by vom Rath (93), 
is 7 ioe This evidently permits only one reduction division in the 
Weismannian sense. Vom Rath and Weismann are therefore inconsistent 
when they hold to a longitudinal splitting in the spirem condition, and 


yet consider both maturation divisions as reductions. If the Vierer- 
gruppen have the formula ie we there are but two sorts of ids, a and 


6, and it is simply impossible to get more than one reduction division. 
a 
Ba 
this is by Weismann’s own definition an equation division, and only 
when these two cells become by division the four ultimate products of 
maturation a, b, a, b, can we speak of a reduction. 

Hicker at first considered both divisions as reductions (’92), but 
later (93) he rightly came to the conclusion that the longitudinal 
splitting in the spirem stage was a preparation for one division, and 
that the final separation of the sister elements thus produced consti- 
tutes an equation division, —a ‘modified equation division,” he calls 
it, because the splitting, which ordinarily occurs at the equator of the 
spindle is here precociously introduced in the spirem condition. 


If from the nucleus oe arises by division two nuclei, Hl and 


24 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


According to my interpretation of the Vierergruppen in Calopte- 
nus, the formula would be ar . Both the divisions following the 


formation of a Vierergruppe would therefore be reductions, and it would 
be quite immaterial whether the first di- 
vision gave rise to two cells ab and ed, 
or to the two cells ac and bd. In Calop- 
tenus the rings may be placed upon the 
spindle equator in either of the two posi- 
tions represented in Diagrams 1 and 2. 
This offers, perhaps, an explanation and 
reconciliation of the contradictory views 
of Henking, Hertwig, Hiicker, and others. 
As has been said, Henking holds that the 
first division is a reduction division, and 
the second an equation division, while most authors make the first an 
equation, and the second a reduction division. Henking (’91) did not, 
in his Pyrrhocoris paper, recognize the existence of Vierergruppen 
as a regular stage in maturation ; but I feel justified by his Figure 20 
in believing that they were really present in Pyrrhocoris, just as in 
Gryllotalpa, Caloptenus, etc. Now, supposing the proper formula for 


the Vierergruppen to be 1 i , why might it not happen in dif- 
ferent nuclei, or in different chromatic groups of the same nucleus, that 
one group should divide thus, — and another thus, A Henking 


must assume that all the groups are arranged on the spindle so as to 
separate the non-identical idants by the first division. Hicker says : 
“In der zwetten Richtungstheilung erfolgt dann die definitive Trennung 
der nichtidentischen Idantenpaare.” 

The following diagrams may illustrate these positions : — 


aa a b aa a $i 
I), a ee oS eae ee 235 
e@ @ e@ 6 @e ee? 
6 6 ru NG 6 6b a $b} 


Diagram I. illustrates Henking’s view of the first division interpreted 
according to the scheme of the Vierergruppen. All the groups would 
thus suffer reduction division. 


WILCOX: SPERMATOGENESIS. 25 


Diagram II. serves to represent the view of Hicker, Weismann, vom 
Rath, and others. ‘The first division is here seen to be an equation di- 
vision. But none of these authors has offered any reliable criterion by 
which we may judge whether it is the “sister idants” or the “ non-identi- 
cal idants”’ that are separated by the first division. They have presented 
no satisfactory evidence that in the same nucleus all groups undergo either 
a reduction division or an equation division. How are we certain that 
one group does not undergo a reduction division at the same time that 
another in the same nucleus passes through an equation division? This 
possibility, as shown in Diagram III., is not excluded. Hicker (92, 
Fig. 22, and ’93, Fig. 116) believes he has seen two examples where the 
non-identical idants were still in connection with each other after sepa- 
ration of the sister pairs in the formation of the first polar globule, but 
the figures are rather unsatisfactory. 

This discussion will, I hope, have made one thing clear: the abso- 
lute necessity of a knowledge of the origin of the Vierergruppen, in 
order to a proper interpretation of the reduction question. If Brauer’s 
account of the origin of the Vierergruppen be correct, there can be 
no reduction. If Hicker and vom Rath have rightly described their 
origins, there is one, and only one, reduction. If my description of the 
ring formation be accurate, there may be two reductions. I am quite 
willing to grant that, as Brauer maintains, precocious preparations for 
both divisions are made in the prophases of the first spermatocyte di- 
vision. But Brauer maintains an origin for the groups of four, which 
determines that each group shall consist of four identical elements, and 
thus does away with Weismannian reduction, while I contend that, 
owing to the manner of their origin, all four elements may be differ- 
ent or unlike one another, and therefore that both divisions may be 
reductions. 

The fate of a Vierergruppe, according to the four views mentioned, 

may again be brought together in diagrammatic form for comparison. 


Brauer :— 
“ a aa a\a aes 
a aa a\a al\a 
Henking : — 
a aa ane BE 
b bb bb AL 


26 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


Vom Rath, Hacker : — 


a aa a\a ca fas 
b b b b| 6} bib 
My view: — 
a\b 
ae ae ale 
cd eal ae 
Sr ene aye 
cd 


As to the question of reduction in the two maturation divisions, only 
four suppositions can be made: (1) the first division only is a reduc- 
tion; (2) the second only is a reduction; (3) both are reductions ; 
(4) neither is a reduction. As we have now seen, all these suppositions 
have been made by different authors. We may well say with vom 
Rath, that “eine allgemein befriedigende Losung ist nur dann méglich 
wenn die Autoren sich zuerst ttber den Begriff ‘ Reduction’ vollig 
geeinigt haben.” 

The assertion of Hiicker and vom Rath (’93), that the resting stage 
which immediately precedes the first maturation division is sometimes 
omitted, has intimate connection with the question of reduction, and its 
meaning, it must be admitted, is not yet entirely explained. The ac- 
counts of Hiicker and vom Rath are too meagre to allow a detailed 
comparison of the processes in cases where there is no “ Keimblas- 
chenstadium ” with those in which it exists. Perhaps we can never 
settle definitely the question of reduction, but material for its solution 
should be sought in a careful determination of the origin and meaning of 
the Vierergruppen. 


CAMBRIDGE, May 16, 1894. 


/ 
WILCOX: SPERMATOGENESIS. ya 


POSTSCRIPT. 


In the Bulletin of the College of Agriculture of the Imperial Univer- 
sity of Japan has recently appeared a paper by Kametaro Toyama (’94), 
“On the Spermatogenesis of the Silkworm.” The author has arrived 
at conclusions which in part agree with my own, but in part are quite 
different. 

Like myself, Toyama was unable to find any longitudinal splitting of 
the chromatic thread in the prophases of the first spermatocyte division. 
He gives the following account of the complicated series of movements 
of the chromatin during the prophases: “A nucleolus is generally seen 
in the network of linin and chromatin. . . . Most of the chromatin 
granules become collected to one side of the nucleus and form an irreg- 
ular mass, ... become again separate from each other and arrange 
themselves along the radiating linin fibres, and the skein stage is thus 
obtained. . . . The chromatin granules scattered in the nucleus 
become again collected in the centre of it, and present an irregular mass 
as before... . In a still later stage the chromatin granules again 
commence to separate from one another. ... A little before the 
appearance of the centrosomes in sperm-mother-cells the chromatin 
granules . . . gradually collect here and there and assume ring-shaped 
structures.” 

Unfortunately the author presents no satisfactory evidence for this 
series of changes. He may have seen all the stages which are enumer- 
ated above, but he gives no proof that they succeed one another in the 
order he has stated. In the earliest prophases Toyama finds the chro- 
matin in nearly the same condition in which I find it in Caloptenus, and 
just before the first maturation division he finds the chromatin arranged 
in quadrivalent rings. The progress toward the ring stage is, according 
to his account, twice interrupted by retrogressive processes. One can- 
not easily conceive the purpose of these complications, and the evidence 
for such an hypothesis could never be conclusive without direct observa- 
tion of the process in the living condition. I know no reason why we 
might not arrange Toyama’s Figures 23-43 in one continuous series. 
Ail stages represented in those figures are very young, and the numerous 
intermediate stages between them and Figure 44 are not shown. The 
concentrated condition of the chromatin seen in the author’s Figure 30 
seems to me due to bad preservation. 

I disagree entirely with Toyama as to the processes in the maturation 


_ 28 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


divisions. Toyama thinks that the Vierergruppen break up into their 
four elements, and that these arrange themselves around the equator 
of the spindle in a single row. He has figured spermatocytes with seven 
Vierergruppen (Figs. 44, 45), and he tells us that there are 28 sep- 
arate chromosomes on the spindle of the first maturation division. We 
must seek his proof of this statement in his Figures 50-52. ‘These seem 
to me to present seven quadrivalent rings, rather than 28 single chromo- 
somes. In his Figure 50 Toyama has represented only 7 chromatic bodies 
on the spindle. Are we to suppose that there were 21 on the other side 
of the spindle? Our author has given no equatorial view of the spindle 
in which simple spherical chromosomes are arranged in one row. His 
Figures 50 and 51 represent what he has considered stages in the trans- 
verse division of the simple chromosomes. But I have seen in Calop- 
tenus chromatic rings in such a position as to give exactly the same 
appearance. In Caloptenus there is no transverse division of the chro- 
mosomes in the maturation divisions, The ring does not break up into 
four simple chromosomes before the period of metakinesis. The separa- 
tion of the ring into its four constituent elements takes place upon the 
equator of two successive maturation spindles. On the first spindle 
each ring is separated into two dumb-bell figures. On the second 
spindle each dumb-bell divides into its two simple chromosomes. Both 
of these divisions are effected by a breaking of the linin fibres between 
the chromosomes, not by division of the chromosomes themselves. 

Toyama’s description of the origin of the ‘“Nebenkern” from the 
remains of the interzonal filaments of the last spermatocyte division is 
essentially the same as I have given for Caloptenus, but he has followed 
its fate in the spermatozo6n farther than I was able to do. His “ mito- 
soma” may be identical with the body which I find in the neck of the 
spermatozo6n of Caloptenus. 


January 19, 1895. 


WILCOX : SPERMATOGENESIS. 29 


BIBLIOGRAPHY. 


Baranetzky, J. 
90. Die Kernteilung in den Pollenmutterzellen einiger Tradescantien. Bot. 
Zeit., Jahrg. XXX VIII. p. 241. 
Blochmann, F. 
'87. Ueber die Richtungskorper bei Insecteneiern. Morph. Jahrb., Bd. XII. 
; p. 544. 
Born, G. 
94. Die Structur des Keimblaschens im Ovarialei von Triton teniatus. Arch. 
f. mikr. Anat., Bd. XLIII. p. 1. 
Boveri, T. 
90. Ueber das Verhalten der chromatischen Kernsubstanz bei der Bildung 
der Richtungskorper und bei der Befruchtung. Jena. Zeit., Bd. XXIV. 
p. 314. 
Brauer, A. 
’'93. Zur Kenntniss der Spermatogenese von Ascaris megalocephala. Arch. 
f. mikr. Anat., Bd. XLII. p. 153. 
Brauer, A. 
94. Zur Kenntniss der Reifung des parthenogenetisch sich entwickelnden 
Hies von Artemia salina. Arch. f. mikr. Anat., Bd. XLIIT. p. 162. 
Carnoy, J. B. 
’85. La cytodiérése chez les Arthropodes. La Cellule, Tom. I. p. 191. 
Carnoy, J. B. 
’86. La cytodiérése de lceuf. La vésicule germinative et les globules polaires 
de l’Ascaris megalocephala. La Cellule, Tom. II. p. 1. 
Carnoy, J. B. 
’86*. Les globules polaires de l’ Ascaris clavata. La Cellule, Tom. ITI. p. 247. 
Flemming, W. 
’°87. Neue Beitrage zur Kenntniss der Zelle. Arch. f. mikr. Anat., Bd. XXIX. . 
p. 389. 
Flemming, W. 
’89. Amitotische Kernteilung im Blasenepithel des Salamanders. Arch. f. 
mikr. Anat., Bd. XXXIV. p. 437. 
Field, G. W. 
93. Echinoderm Spermatogenesis. Anat. Anzeiger, Jahrg. VIII. p. 487. 


30 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


Frenzel, J. 
91. Zur Bedeutung der amitotischen Kernteilung. Biol. Centralblatt, Bd. 
XI. p. 558. 
Frenzel, J. 
91", Die nucleolare Kernhalbierung. Biol. Centralblatt, Bd. XI. p. 701. 
Geberg, A. 
91. Zur Kenntniss des Flemmingschen Zwischenkorperchens. Anat. Anzei- 
ger, Jahrg. VI. p. 628. 
Guignard, L. 
’91.. Nouvelle étude sur la fécondation. Ann. de Sci. Nat. (Bot.), Tom. 
XIV. p. 163. 
Hacker, V. 
92. Die Hibildung bei Cyclops und Canthocamptus. Zool. Jahrbiicher, Aoth. 
EiAnat. Bde Ve, 211s 
Hacker, V. 
92% Die heterotypische Kernteilung in Cyclus der generativen Zellen. Ber. 
naturf. Gesell. Freiburg, Bd. VI. p. 160. 
Hacker, V. 
’93. Das Keimblaschen, seine Elemente und Lageveranderungen. I. Ueber 
die biologische Bedeutung des Keimblaschenstadiums und iiber die Bildung 
der Vierergruppen. Arch. f. mikr. Anat., Bd. XLI. p. 452. 
Heidenhain, M. 
92. Ueber Kern und Protoplasma. Kolliker’s Festschrift, p. 111. 
Henking, H. 
90. Die ersten Entwicklungsvorgange in den Hiern der Insecten. Zeit. f. 
wiss. Zool., Bd. XLIX. p. 563. 
Henking, H. 
91. Erste Entwickelungsvorgiange in den Hiern der Insecten. Zeit. f. wiss. 
Zool., Bd. LI. p. 685. 
Henking, H. 
92. Die ersten Entwickelungsvorgainge in den Hiern der Insecten. Zeit. f. 
wiss. Zool., Bd. LIV. p. 1. 
Henneguy, L. F. 
*91. Nouvelles recherches sur la division indirecte. Jour. Anat. et Phys., 
Tom. XXVII. p. 397. 
Hermann, F. 
’89. Beitrage zur Histologie des Hodens. Arch. f. mikr. Anat., Bd. XXXIV. 
p- 58. 
Hertwig, O. 
90. Vergleich der Ei- und Samenbildung bei Nematoden. Arch. f. mikr. 
Anat., Bd. XXXVI. p. 1. 
Kultschitzky, N. 
’88. Die Befruchtungsvorginge bei Ascaris megalocephala. Arch. f. mikr. 
Anat., Bd. XXXI. p. 567. 


WILCOX: SPERMATOGENESIS. ol 


Kultschitzky, N. 
’88". Ueber die Eireifung und Befruchtungsvorginge bei Ascaris marginata. 
Arch. f. mikr. Anat., Bd. XXXII. p. 671. 
La Valette. (See St. George, La Valette.) 
Léwit, M. 
91. Ueber amitotische Kernteilung. Biol. Centralblatt, Bd. XI. p. 518. 
Lukjanow, S. M. 
’89. Linige Bemerkungen iiber sexuelle Elemente beim Spulwurm des Hun- 
des. Arch, f. mikr. Anat., Bd. XXXIV. p. 397. 


Meves, F. 
91. Ueber amitotische Kernteilung in den Spermatogonien des Salamanders, 
und Verhalten der Attraktionssphare bei derselben. Anat. Anzeiger, Jahre. 
VI. p. 626. 


Moore, if Ess 
93. Mammalian Spermatogenesis. Anat. Anzeiger, Jahrg. VIII. p. 683. 


Platner, G. 
'86. Ueber die Entstehung des Nebenkerns und seine Beziehung zur Kern- 
teilung. Arch. f. mikr. Anat., Bd. XX VI. p. 343. 


Vom Rath, O. 
‘90. Ueber eine eigenartige polyzentrische Anordnung der Chromatins. Zool. 
Anzeiger, Jahrg. XIII. p. 231. 
Vom Rath, O. 
91. Ueber die Bedeutung der amitotischen Kernteilung im Hoden. Zool. 
Anzeiger, Jahrg. XIV. pp. 331, 342, and 355. 


Vom Rath, O. 
'91". Ueber die Reduction der chromatischen Elemente in der Samenbildung 
von Gryllotalpae Ber. naturf. Gesell. Freiburg., Bd. VI. p. 62. 


Vom Rath, O. 
’92. Zur Kenntniss der Spermatogenese von Gryllotalpa vulgaris Latr. 
Arch. f. mikr. Anat.. Bd. XL, p. 102. 
Vom Rath, O. 
93. Beitrage zur Kenntniss der Spermatogenese von Salamandra. Zeit. f. 
wiss. Zodl., Bd. LVII. p. 97. 
Riickert, J. 
92. Zur Entwickelungsgeschichte des Ovarialeies bei Selachiern. Anat. 
Anzeiger, Jahrg. VII. p. 107. 
Riickert, J. 
'93. Ueber die Verdoppelung der Chromosomen im Keimblaschen des Sela- 
chiereies. Anat. Anzeiger, Jahrg. VIII. p. 44. 
Sabatier, A. 
790. De la Spermatogenése chez les Locustides. Compt. Rend. Acad. Paris, 
Tom. CXI. p. 797. 


_ 82 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


St. George, La Valette. 
85. Spermatologische Beitrage. I. Arch. f. mikr. Anat., Bd. XXV. p. 581. 


St. George, La Valette. 
’86. Spermatologische Beitrage. IV. Arch. f. mikr. Anat., Bd. XX VIIL p.1. 
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94. On the Spermatogenesis of the Silkworm. Bull. Coll. Agric. Imp. Univ. 
Japan, Vol. II. No. 3, p. 125. 


Verson, E. 
91. Zur Beurteilung der amitotischen Kernteilung. Biol. Centralblatt, Bd. 
I. p. 556. 
Wagner, J. 
’92. A Review of the Present Condition of the Question as to the Existence 
and Meaning of Fertilization. (Russian.) Rev. de Sci. Nat. St. Péters- 
bourg, pp. 88 and 145. 


Weismann, A. 
‘91. Amphimixis oder die Vermischung der Individuen. Jena: G. Fischer. 


Weismann, A. 
92. Das Keimplasma. Jena: G. Fischer. 


Zacharias, O. 
’87. Neue Untersuchungen iiber die Copulation der Geschlechtsproducte und 
den Befruchtungsvorgang bei Ascaris megalocephala. Arch. f. mikr. Anat., 
Bd. XXX. p. 111. 


Zeigler, H. E. i 
’'91. Die biologische Bedeutung der amitotischen Kernteilung im Tierreich. 
Biol. Centralblatt, Bd. XI. p. 372. 


Zeigler, H. E., und Vom Rath, O. 
91. Die amitotische Kernteilung bei den Arthropoden. Biol. Centralblatt, 
Bd. XI. p. 744. 


EXPLANATION OF PLATES. 


Figures 1-104 (Plates I. and II.) are from Cicada tibicen; Figures 105-244 (Plates 
III-V.) from Caloptenus femur-rubrum. All figures were drawn by means of an 
Abbé camera lucida. The tube was drawn, giving a length of 199mm. Zeiss 
lenses were used in all cases. Figures 14 and 108 were magnified 90 diameters ; 
Figures 7, 8, 12, 13, and 19-22, 870 diameters; Figures 227-229, 251, and 242, 1,080 
diameters; Figures 62-77, 1,400 diameters ; all other Figures, 680 diameters. 


Witcox. — Spermatogenesis. 


PLATE I. 


Cicada tibicen. 


Fig. la-e. Spermatids in various stages of metamorphosis. 


Fig. 2. Spermatid with nucleus Nebeukorper and small red body. 

Fig. 3. Spermatid, tail undeveloped. 

Fig. 4. Spermatid immediately after second division of spermatocytes. 
Figs. 5,6. Spermatids. 

Fig. 7. Spermatogonium with centrosomes near nucleus. 

Fig. 8. Spermatogonium after amitotic nuclear division. Degeneration. 
Figs. 9, 10. Spermatids. 

Fig. 11. Spermatogonia. 

Figs. 12, 13, 21, 22. Stages in development of giant spermatozoa. 

Fig. 14. Longitudinal section of testicular follicle. 


Figs. 15-18, 24-43. Stages in the metamorphosis of spermatids. See also Figs. 44, 
45 (Plate II.). . 

Fig. 23. Part of compartment ¢ of Figure 14. To the left of the connective tissue 
dissepiment are spermatogonia; to the right, below, are spermato- 
cytes of the first order; above, spermatids. 

Figs. 19, 20. Degenerating spermatogonia. Fig. 19 with two extranuclear bodies, 
centrosomes ? 


PLATE. 


ct 
ww. 


WILGOX.- SPERMATOGENESIS 


-B Meisel lith Boston. 


Witcox. — Spermatogenesis. 
t 


PLATE IL. 


Cicada tibicen. 


Figs. 44, 45. Advanced stages in the metamorphosis of spermatids. 

Figs. 46-48. Spermatogonia. 

Figs. 49-61. Spermatocytes, showing different positions of the body I suppose to 
be the nucleolus. 

Figs. 62-77. Metamorphosis of spermatids, < 1,400. 

Figs. 78-103. Metamorphosis of giant spermatids, X 680. 

Fig. 104. Normal spermatids from same microscopic field as Figures 78, 103, 
x 680. 


4h 


EVW del 


WILGOX.- SPERMATOGENESIS. 


&2 
es aS 


“GRD 


_ 


WILcox. — Spermatogenesis. 


PLATE III. 


Caloptenus femur-rubrum. 


Figs. 105-107. Spermatogonia. 

Fig. 108. Longitudinal section of testicular follicle. a, spermatogonia; b, growth 
zone of spermatocytes; c, first division of spermatocytes; d, first stages 
of spermatids ; ¢, later stages of spermatids; f, abortive spermatogonia. 

Fig. 109. Cross-section of vas deferens containing spermatozoa. 

Figs. 110, 111. Spermatocytes in the spirem stage. 

Figs. 112, 113, 117, 118, 122, 123. Spermatocytes in the first division. 

Fig. 114. Spermatocyte of the first order before dumb-bell stage of chromatin. 

Figs. 115, 116. Spermatocytes of the first order in the dumb-bell stage of chromatin. 

Figs. 119-121, 124. Spermatogonia in division. 

Figs. 125, 126, 129. Spermatids just after division of the spermatocytes of the 
second order. 

Fig. 127. Spermatocyte, first division. ‘ 

Fig. 128. Spermatocyte, second division. 

Fig. 180. Spermatocyte before first division. 

Fig. 131. Spermatogonium, polar view of spindle. 

Figs 132-138. Spermatogonia in division. 


B Meisel, lith Boston. 


WILCGOX.- SPERMATOGENESIS. 


as 
> 5 


Witcox. — Spermatogenesis. 


Figs. 139-158. 
Figs. 159, 160. 


Fig. 161. 


Figs. 162, 163. 
Figs. 164-171. 
Figs. 172-176. 


Fig. 177. 


Figs. 178-183. 
Figs. 184-188. 


Fig. 189. 


PLATE IV. 
Caloptenus femur-rubrum. 


Spermatid metamorphosis. 

Spermatocytes, first division. 

Spermatocyte, preparatory to first division. : 

Spermatocytes with chromatin in dumb-bell stage. 

Spermatogonia. 

Spermatocytes, stages in ring formation. 

Spermatocyte, first division. One centrosome is cut away in 
another section. 

Spermatocytes, ring stages. 

Spermatocytes, spirem stages. 

Spermatogonium, tripolar division. 


a 


C 


WILGOX.- SPERMATOGENESIS. 


tt 


Pn, JUL 142 nt 14#4 
: Ss if © 
ae ‘ i] j / 
‘f \j } pred —_ 
é e 3 aa at 
; 
a” 


145 qs 146 147 pr” 149 ‘ 151 
J ao” 9 
j 


B Meisel lith Boston. 


Witcox. — Spermatogenesis. 


Figs. 190, 191. 
Figs. 192-195. 
Figs. 196-200. 
Fig. 201. 

Figs. 202-220. 
Figs. 221-229, 244. 
Figs. 230, 252-256. 
Fig. 231. 

Figs. 237-241. 
Figs. 242, 243. 


PLATE V. 


Caloptenus femur-rubrum. 


Spermatocytes, second division. 
Spermatocytes, first division. 

Spermatid metamorphosis. 

Spermatocyte just before ring condition. 
Degenerating cells. 

Spermatocytes, preparatory to first division. 
Spermatids. 

Spermatocyte, second division. 
Spermatocytes, first division. 
Spermatocytes, ring formation. 


2 it 
195 
I94- 
aN. 
3 , £0 aaa 
193 ff 


| Pte / 
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7 


of the Museum of Comparative Zodlogy 
ss AT ~ HARVARD COLLEGE, 
Weevor xX Vil, No. 2. 


- , 
ON THE EARLY DEVELOPMENT OF LIMAX. 
J 
: al Siyatl Ops Kororp. 
- | | i @ 
es 75 : 
f ner. Orde Eh y 
p . 
oan Ercut PLATEs. 


CAMBRIDGE, MASS., U.S. A.: 
| PRINTED FOR THE MUSEUM. 
—. Aveusr, 1895. 


Bulletin of the Museum of Comparative Zodlogy 
AT HARVARD COLLEGE. 
Vou. XXVII. No. 2. 


ON THE EARLY DEVELOPMENT OF LIMAX. 


By C. A. Kororp. 


With E1cut PLuAtTeEs. 


CAMBRIDGE, MASS., U.S. A.: 


PRINTED FOR THE MUSEUM. 
AvGtstT, 1895. 


ie ke iusto wot en ee 


No. 2. — On the Early Development of Limaz.’ By ©. A. Kororp, 


TABLE OF CONTENTS. 


Page Page 
ieeintroduction . .... . ood D. The Mesoderm .. . 715 
Mee Methods . .. . 3 E. Theoretical Goneideradons wi 
III. Nomenclature ai Spinal Cleavage 40 F. The Cleavage Cavity . . 81 
IV. General Sketch of the Develop- HS -Lysy Oy reoy: bree Syed! 
MGUG ies es) es acgs te 140 Zeeloiteraturema, 3. OL 
WerCleavage . 3... 1... . 644 3. Experimental. . . . 104 
Introductory. . . 44] VI. Blastopore and Gastrulation . 106 
A. Orientation oftlie Bmbrye ANoy || NGlotyeviliimn 5, go) oe Suen tee ealll 
B. Discussion of Cleavage . 46] Literature Cited. . .... =. 112 
C. Literature on Spiral Cleav- Iixplanation of Plates . . . . . 118 

ACN pecan mer |) sa BOG, 


I. INTRODUCTION. 


THE question of the origin and history of the mesoderm, and its rela- 
tion to the body cavity in the Mollusca, is one of prime interest and 
importance. The employment of the mollusk as the type of the 
“ Pseudocoels ” by the Hertwigs (81) in their “ Coelomtheorie ” was 
founded on the non-existence in mollusks of a true body cavity, the 
mesenchymatous nature of the musculature, and the origin of the ner- 
vous system, in part at least, from the mesoderm ; in a word, on the 
nature of the middle germ layer in its origin and later history.” Since 
the publication of this important work many additions have been made 
to our knowledge of the Mollusca. There is a notable agreement among 
later investigators, especially Schmidt (’90), Miss Henchman (’91), and 
Erlanger (91), as to the ectodermal origin of the nervous system 
in this group. Studies in comparative anatomy, particularly of that 
primitive group, the Solenogastres, have led to the general acceptance 
of the view that the pericardium of the Mollusca is the homologue 
of the colom of the “ Enterocoels” of the Hertwigs. This view is 
based upon the relationship of the pericardium to both the sexual 
and excretory systems, embryology however having lent little support 


1 Contributions from the Zodlogical Laboratory of the Museum of Comparative 
Zoology at Harvard College, under the direction of E. L. Mark, No. XLVIII. 
VOL. XXVII. — NO. 2. 3 


36 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


to the view until the recent important work of R. von Erlanger 
(91) upon Paludina, in which he demonstrates that the mesoderm 
originates by a pair of ventral out-pocketings, and that there is 
a close connection, though not continwity, of the paired fundaments of 
the pericardium with these out-pocketings. As yet his results are iso- 
lated, and the hope that a similar origin of the mesoderm might be found 
in other Mollusca is unfulfilled, at least so far as other investigators 
are concerned. 

The generally vague and often contradictory results obtained by vari- 
ous investigators of molluscan embryology concerning the origin of the 
mesoderm have made very apparent the need of careful and detailed 
work along the lines laid down by Rabi (’79), Blochmann (’81), Wilson 
(92), Conklin (92), and Heymons (’93). It is only in the light of 
such work as this that a classification of sweeping import, like that of 
the Hertwigs’ ‘“Coelomtheorie,’ can find its final justifivation, if it 
has one. 

It was with a view of adding something to our knowledge of the 
details of this subject that the work in hand was undertaken. The 
pursuit of this has led me unavoidably into the study of the cleavage, 
and to a certain extent into the field of cell lineage. 

Limax seemed for many reasons to be a desirable object for my inves- 
tigation. The adults are readily procurable, and an abundant supply of 
eggs whose age is approximately known can be obtained from animals 
kept in confinement. The absence of a large amount of nutritive yolk 
leaves the eggs sutliciently translucent for examination in toto, and 
makes section-cutting feasible, though the smallness of the eggs renders 
their orientation in certain stages difficult. 

My work was begun in the fall of 1892, at the suggestion of Dr. E. L. 
Mark, to whose kindly interest and guidance I owe very much. Agrio- 
limax agrestis L. was the species chosen for the work, as the adults are 
abundant at that time of the year about piles of rubbish and stone 
heaps, — in fact, wherever decaying vegetable matter and moisture afford 
food and a suitable retreat. After the last of November, a supply of 
adults can generally be secured in greenhouses. 

The eggs of other Limaces can also be collected in the same localities, 
and as those of Agriolimax agrestis are not readily distinguished from 
some of them, recourse was had to eggs of known parentage only. 


KOFOID: DEVELOPMENT OF LIMAX. 37 


Il. METHODS. 


The most successful method of keeping the animals in captivity was 
found to be as follows. A tin box with proper ventilation is filled to 
the depth of one inch with clean sand, which formsa suitable substratum 
for the retention of moisture. On this is laid down a sheet of moss, to 
whose under surface the earth still adheres. The leaves of the common 
_ plantain furnish acceptable food, and, when this is no longer available, 
fresh cabbage leaves and apple parings can be used. The eggs, which 
seem more often to be laid at night, are found in clusters in the soil, or 
‘eunningly packed away in the moss itself. The rate of development is 
such at the ordinary temperature of the laboratory that the eggs col- 
lected in the morning will generally be found to have already reached 
the early stages of cleavage, while gastrulation progresses during the 
second day, and is completed early in the third. During the first week 
of captivity the slugs furnish eggs in great abundance; but after that 
time the number diminishes and the quality deteriorates so rapidly that 
it is imperative that a new colony be secured. Abnormalities in the 
living egg show themselves in the early stages by a loose assemblage of 
the cells, and the increasing opacity of the embryo. 

_ Before hardening the embryo, it is necessary to free it from the en- 
velopes and albumen which surround it. As the eggs of Agriolimax 
agrestis are much smaller than those of Limax maximns, it was not 
possible to employ the method described by Miss Henchman (’91) for 
shelling the eggs. But by inserting two fine cambric needles in one 
holder, so that the distance between the points is less than the diqmeter 
of the unshelled egg, it is possible to hold the egg between these two 
_ needles and pierce it by a third. A quick shear-like cut with the third 
needle against one of the other two tears open one side of the egg and 
allows the albumen and the ovum to escape from the envelopes. It is 
very desirable not to entangle the embryo in the viscous matter between 
the inner and outer envelope, for it is almost impossible to remove this 
when it is once attached to the embryo. The albumen interferes with 
section-cutting and obscures whole preparations, so that it is necessary 
to remove it entirely. This for a long time presented a most serions 
obstacle to my work. Washing off the albumen with water is a very 
slow and tedious process, and not always successful. Some of the eggs, 
after treatment with Merkel’s or Flemming’s fluid for a short time, were 
washed with hypochlorite of soda to rid them of the albumen. The 


38 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


difficulty of using this lies in the necessity of stopping the action of 
the hypochlorite before it attacks the ovum. It frees the eggs of 
the albumen, and does not interfere with staining, but the proportion 
of eggs destroyed in the process is very great. When the eggs are 
thrown into weak chromic acid (one fourth to one third per cent) or 
weak Merkel’s fluid, the albumen is coagulated, and if the exposure is 
properly timed, the egg can sometimes be freed from its envelopes. It 
is difficult to get good series of sections of eggs hardened in their enve- 
lopes, or but partially freed from them. The process of dehydrating and 
embedding renders the albumen so hard and brittle that it breaks into 
bits when a microtome knife strikes it, and generally the whole section 
becomes shattered. This is especially true of eggs killed in any of the 
chromic fluids. The method of Schmidt (90) for the later stages of 
Limax was employed with success for the early stages. The eggs are 
thrown into a saturated aqueous solution of corrosive sublimate, and as 
soon as they have become opaque they are washed in water, shelled, and 
freed from the coagulated albumen by a gentle stream of water from a 
pipette. There is less danger of distorting or destroying the egg in the 
process of shelling by this method than by any other ] have employed ; 
the disadvantages are that one is limited to this single killing reagent, 
and that it is often difficult to remove all the albumen. 

The method which gives by far the best results is as follows. The 
living eggs are placed in normal salt solution (0.75 per cent), in which 
they are at once shelled, and then freed from the albumen by washing 
them in the salt solution, which is dropped upon them from a pipette. 
The operation is carried on in large glass dishes, resembling watch- 
glasses, but provided with flat polished bottoms, which are placed upon 
a black tile ; this renders the eggs visible to the naked eye. The salt 
solution dissolves away the albumen, leaving the egg entirely free. It 
can then be transferred to any desired killing reagent by the use of a 
capillary glass tube. It is advisable to shorten the exposure in the salt 
solution as much as possible, for nuclear conditions are somewhat altered 
by its action. Eggs which have lain in it for ten minutes have their 
nuclear membranes much distended, and the chromatin, gathered into a 
homogeneous mass at the centre of the nucleus, surrounded by a clear 
region of nuclear sap. It is possible, however, by this method to obtain 
eggs whose nuclear conditions do not seem to be in the least changed. 

For killing reagents the following were used: picro-sulphuric ; 
picro-sulphuric with a few drops of one per cent osmic added to it 
(Erlanger, 91); Perenyi, followed by five per cent alum water ; Whit- 


KOFOID: DEVELOPMENT OF LIMAX. on 


man’s Merkel; Fol’s modification of Flemming’s chrom-osmic-acetic, 
either alone or followed by Whitman’s Merkel. By far the most satis- 
factory results were obtained by subjecting the eggs to the action of 
Fol’s modification of Flemming’s mixture for one minute and transfer- 
ring them at once to Orth’s picro-carminate of lithium. The eggs were 
allowed to remain in the stain twelve to twenty-four hours, and were 
then decolorized with acidulated alcohol until the cytoplasm retained 
but a slight tinge of red. Rapid decolorization with ninety per cent 
alcohol plus five per cent hydrochloric acid gave very good results. 
The eggs when properly decolorized have cell boundaries and nuclear 
membranes sharply marked, and the chromatic elements of the nucleus 
remain a deep red. Asters show plainly, but centrosomes are not 
stained. Eggs killed in Flemming’s fluid and afterwards bleached by 
chlorine, or those killed in Merkel’s fluid, are satisfactorily stained in 
Mayer’s HCl-carmine. These also must be thoroughly decolorized. 
Kgs killed in corrosive sublimate were stained in alum-carmine or 
Czokor’s cochineal, but the best results after this killing agent were 
obtained by the addition of a drop of Delafeld’s hzematoxylin to slightly 
acidulated water in which the eggs had been placed after hardening in 
alcohol (Conklin, 92). This is especially valuable for the demonstra- 
tion of astroccels in the early stages of cleavage. Satisfactory results 
were not obtained on whole preparations with Heidenhain’s iron-hema- 
toxylin or Henneguy’s method with permanganate of potash and safra- 
nin. The first, however, gives very good results with sections. 

The processes of killing, hardening, staining, and clearing were carried 
on in watch-glasses. Capillary glass tubing was found to be very con- 
venient for transferring individual eggs when such transfer was necessary. 
Turpentine, xylol, or cedar oil was used as a clearing agent. Eggs can 
be kept without harm for a long time in turpentine evaporated down to 
a waxy consistency, or in xylol to which soft parafine has been added. 
If the xylol is allowed to evaporate, it leaves the eggs embedded in the 
soft parafine, which can be redissolved by fresh xylol without harm even 
to these very delicate objects. 

The eggs were studied in the clearing agent under a cover-glass placed 
on glass rollers made of bits of capillary tubing. This allows the use of 
high-power objectives and the orientation of the embryo in any desired 
position for a camera drawing. When permanent preparations were 
desired, they were mounted in xylol-balsam or a solution of dammar in 
cedar oil. By the addition of a drop of xylol to the margin of the cover- 
glass, the mounting medium is sufficiently softened to allow the cover- 


40 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


glass to be moved upon the rollers, and the egg oriented as desired, even 
after the slide has stood for months. 

The method of embedding and orienting preparatory to it de- 
scribed by Dr. Woodworth (’93) proved to be very valuable. The 
ordinary method of orienting in melted parafine on the warm stage 
with the aid of a lens was also employed. Sections were cut 6.67 p 
in thickness, and reconstructions of many stages were made in wax 
on a scale of three hundred diameters. Transverse, sagittal, and frontal 
sections were cut; though it was not always possible to orient the 
embryo exactly, the reconstructions revealed the direction of the sections 
in cases where there was doubt. Sagittal sections are more readily 
interpreted than the others, for in them the cells of the different germ 
layers are shown in the same section in such relations as to be more 
easily recognized than in sections in other planes. 

In the discussicu of sections the following orientation is used. The 
end called anterior is the one toward which the growing invagination is 
directed. At the time of gastrulation it is the larger end of the embryo. 
The opposite end is the posterior, and is marked at the stage preced- 
ing gastrulation by a greater thickness than the anterior end, due to 
the presence of the mesoderm. In the early stages of gastrulation 
the broader and shallower end of the blastopore lies anterior. At the 
completion of gastrulation the contracted remnant of the blastopore 
occupies a terminal position at the posterior pole. The chief axis is the 
antero-posterior one. The ventral surface is marked in the blastula by a 
greater convexity than the dorsal, but during the period of gastrulation 
by the growing invagination. Sections are called sagittal that are 
parallel to the plane which coincides with both chief and dorso-ventral 
axes; frontal, those that are perpendicular to the dorso-ventral axis ; 
transverse, those that are perpendicular to the chief axis.. 


Ill. NOMENCLATURE OF SPIRAL CLEAVAGE. 


The earliest full discussion of spiral cleavage occurs in Blochmann’s ad- 
mirable work upon Neritina (81). Fol (’75 and ’76) had described the 
early stages in the cleavage of the Pteropods and the Heteropods, and 
Rab] (79) the cleavage of Planorbis ; but neither had entered into a full 
discussion of the lineage of theicells or the spiral character of the cleavages 
with which he was dealing. In Neritina the cleavage is unequal, and 
at the formation of the first set of micromeres we have the appearance of 


KOFOID: DEVELOPMENT OF LIMAX. 41 


a small protoplasmic mass budded off from a larger mass. This concep- 
tion of the cell division —the derivation of a small part from a large 
part — dominated Blochmann’s nomenclature both of cells and of spirals. 
Accordingly, we find him designating a large mass of protoplasm, both 
before and after the small mass is budded off from it, by the same name, 
So also, when he comes to compare the spiral with the motion of the 
hands of a clock, he regards the small cell as moving away from the large 
cell, and designates the spiral accordingly. Other investigators of spiral 
cleavage — Lang (’85), Conklin (’91 and ’92), Wilson (’92), Heymons 
(93), Lillie (93) — have, like Blochmann, dealt with forms presenting a 
greater or a less inequality in cleavage, and have found it convenient to 
employ the system inaugurated by Blochmann for their nomenclature 
of cells and spirals. There has arisen in the usages of these various 
authors, however, considerable confusion in the detailed application of 
their nomenclatures to this basis of reference. Indeed, as I have pointed 
out in a previous paper (Kofoid, ’94), an author is not always able to 
avoid inconsistencies. This state of affairs is confusing and extremely 
annoying to the student who wishes to make a comparative study of 
cell lineage. However much the introduction of a new system of nomen- 
clature is to be deplored, it seems to be justified for the following reasons. 
Cell lineage deals primarily with the descent and fate of cells, and is most 
conveniently traced by following the history of their nwclec ; it is only 
secondarily concerned with the amount of yolk or protoplasm in the 
cells. The founding of a system of nomenclature, therefore, upon the 
relative sizes of cells, ignores wholly this fundamental proposition, and 
substitutes a basis of varying and uncertain nature. Furthermore, this 
system has caused the introduction, perhaps not necessarily,.of the 
custom of designating cells of different generations by identical names ; 
thus A may be a cell of any one of a half-dozen different generations. 
In this, too, the principle of descent is ignored. 

Finally and principally, the basis hitherto employed affords no solid 
ground whatever for comparisons, for it gives no logical method to be 
employed in cases of equal cleavage ; and its application must vary with 
the varying distribution of the large cells in different species of animals. 
Thus it comes about that ‘ homologous” cells, i. e. those of identical 
descent, must according to this system be named differently in differ- 
ent animals. It may be that the system as applied by these authors 
does furnish a means, readily grasped by the eye and the mind, of fol- 
lowing the lineage in the particular form studied ; but so long as it fails 
to form a basis for comparison, it is open to serious objection. It was 


42 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


with a view of meeting this objection and suggesting a suitable basis of 
comparison that I proposed, in a paper (’94) to which I must refer the 
reader for a detailed description, an entirely new principle of nomencla- 
ture of both cells and spirals. This is a strictly genealogical system, 
giving to each cell in the line of descent a separate designation, one de- 
termined moreover by the constant spatial relations common to all eggs, 
and not by the inequality of the cleavage characteristic of individual 
species. 

The system presupposes the division of the egg into four quadrants 
designated a, b, c, d, placed in the order in which the hands of a clock 
move. These quadrants are occupied by the four blastomeres, the 
quartet, of the third generation. When this quartet, or any other quar- 
tet of the later stages, divides, forming two quartets, each cell is desig- 
nated as follows: (1) by a letter indicating the quadrant, as, e. g., a; 
(2) by a first exponent indicating the generation, a*, a4, etc. ; (3) by a 
second exponent indicating the posztion of the quartet with reference to all 
other quartets of the same generation, potential or actual, the quartets 
being numbered from the vegetative toward the animal pole, as a*, at, 
etc. Thus the cell a’ divides, forming a*! and a*?; in the second ex- 
ponent the odd one being always given to the cells of the quartet which is 
nearer the vegetative pole, and the even to those of the quartet nearer the 
animal pole. I have previously described (’94) the simple and constant 
manner in which the designation of the daughter cells can in every case 
be derived from that of the mother cell. 

It may be well to call attention here to the significance of this system 
of nomenclature. It designates cells as they might be named in the 
simplest possible mathematical and mechanical conditions of a cleaving 
egg, i.e. equal, regular cleavage pervading all the cells of a generation 
at the same time. In such a case we should have all the quartets of a 
generation actually present and numbered in the regular succession of 
their position from the vegetative to the animal pole. The possibility 
of referring all forms of spiral cleavage to such a simple type is obvious: 
and the advantage, if not indeed the necessity, of such a reference as a 
basis of comparison is equally apparent. The fact that in the applica- 
tion of this system the exponents have little or no significance, or are 
even misleading, as to the actual number of quartets present between a 
given quartet and the vegetative pole, is thus no obstacle, when once 
the real significance of the system is understood. In fact, it is rather 
an advantage that the regions of most rapid growth in the embryo are 
thus prominently designated. There are doubtless objections that 


KOFOID : DEVELOPMENT OF LIMAX. 43 


will be encountered in the application of the system. After the ninth 
generation of cells the exponents become exceedingly cumbersome, but 
this is an objection which applies to all other systems as much as, or to 
even a greater extent than, to this ; and it may perhaps in many cases be 
avoided in the later stages by the introduction, for teloblasts and their 
progeny, of subordinate dichotomous systems based on combinations of the 
numerals 1 and 2, as introduced by Chabry (’87), and later adopted by 
Wilson (’92) in his subordinate systems. It is also true that the system, 
as proposed, does not optically differentiate the macromeres and the 
primary, secondary, and tertiary micromeres where it is desirable to 
distinguish these groups or their immediate descendants. This however 
is readily accomplished by the use of differential type, or even by other 
letters of the alphabet than a, 6, c, d, but used in the same order. 

There seems to be no doubt that this system can be applied wher- 
ever it is possible to divide the cleaving egg into equivalent quadrants, 
and thus to distinguish quartets of cells. I have myself applied it to 
the spiral cleavage described for various forms (see review of the litera- 
ture), and my friend, Mr. Castle, has applied it successfully to the 
bilateral cleavage of Ciona and to that of Clavelina as described by Van 
Beneden et Julin (’84). 

To make this system available in all cases, it is only necessary to 
apply the second exponent in a constant manner with reference to some 
spatial relations; e. g. in the case of Tunicate cleavage, with reference 
to the sagittal and transverse planes, starting in all cases from the 
vegetative pole. 

In conclusion, it hardly needs to be suggested that the generation 
basis of comparison is about the only one that can be employed between 
the various types of cleavage ; and I would add that it promises to be 
useful in the discussion of precocious development. 


IV. GENERAL SKETCH OF THE DEVELOPMENT. 


Limax has spiral cleavage of the typical form, the spirals alternating 
in successive cell generations, right spirals resulting in the even genera- 
tions and left spirals in the odd. The mesoderm is derived from the left 
posterior quadrant, and, as in Nereis, Umbrella, Crepidula, and Unio, 
the first mesoblast cell is d‘*. An ephemeral, recurrent cleavage cavity 
appears at the two-cell stage, and recurs as late as the completion of the 
period of gastrulation. This cavity is excretory in function, and is 


44 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


induced by the environment of the egg. The primary mesoblast divides 
bilaterally, ultimately sinks below the general level, and forms two 
bilaterally placed mesodermal bands extending anteriorly. Their forma- 
tion precedes and accompanies gastrulation, no lumina appearing at any 
time within them. The blastopore is at first broad and shallow, but it 
gradually deepens at the anterior end, and disappears from the posterior 
margin anteriorly, forming an elliptical pit on the median ventral surface. 
By a rapid growth in the latero-anterior lips of this pit, accompanied by 
an accumulation of mesoderm in these regions and a general readjust- 
ment of the axes of the embryo, the opening leading into the archenteron 
assumes a position at the former posterior margin of the blastopore. 
This remnant of the blastopore comes to lie in the anal region; the 
roouth breaks through at a later period upon the ventral surface of the 
embryo. 


V. CLEAVAGE. 


Introductory. 


The cleavage of the egg of Limax takes place with considerable rapidity. 
The eggs are generally laid, in captivity, during the night, and in the 
morning one finds stages from the one-cell to the sixteen- and occasion- 
ally the twenty-four-cell stage. By six o’clock in the evening these 
eggs have reached the stages of twenty-four to forty or more cells. 
Gastrulation begins during the second day, and is completed on the 
third day. There is, however, much variation even in a lot of eggs 
found in one mass, and evidently laid by one individual. These dif- 
ferences may possibly be due to differences in the time of fertiliza- 
tion. Temperature exercises a profound influence on the rate of cleay- 
age. Eges about to pass into the twenty-four-cell stage at 6 P. M. were 
placed over night in a temperature a few degrees above freezing, and 
were found to have just reached that stage at 8.30 the next morn- 
ing, and, though restored to the temperature of the laboratory, they did 
not progress to the next cleavage until 2 p.m. There are a few “ stages ” 
in the cleavage that are well marked, i. e. periods of apparent inactivity 
in which the egg continues for some time. These are the the two-, four-, 
eight-, sixteen-, twenty-four-, forty-four-, and sixty-cell stages. The 
periods alternating with these are marked by mitotic conditions in all 
or a part of the cells of the egg. 

The animal pole of the mature and undivided egg is marked by the 
presence of two polar globules. These generally differ in size, the more 


KOFOID : DEVELOPMENT OF LIMAX. 45 


distant, i. e. the first, being the larger. In stained preparations the 
larger one often contains a distinct nucleus with nuclear membrane and 
chromatic granules (Plate III. Figs. 20, 21). In the case figured here 
the two globules are closely applied to the surface of the egg. In the 
majority of instances, however, they lie at some distance from the egg in 
the albumen, and in the living egg often seem to have no connection 
whatever with the vitelline surface. Thus it happens that the polar 
globules are removed with the albumen in by far the larger part of the 
eges shelled. A phenomenon observed occasionally in the later stages of 
the living egg is the increase in size of one of the polar globules and its 
subsequent collapse (Plate I. Figs. 9-11). In one case the globule 
reached a diameter half that of the egg itself. This is apparently 
caused by the absorption of fluid from the albumen, and in the case 
noted was followed by a collapse and a return to the normal size and 
shape. The eggs of Agriolimax agrestis vary a great deal in size, the 
limits being from 80 u to 160 win diameter. The average size is about 
100-110 pw. 


A. Orientation of the Hmbryo. 


In my treatment of the subject the orientation employed by Wil- 
son (92), Conklin (92) and Heymons (93) is followed. The first 
cleavage plane is transverse, the second sagittal, in relation to the future 
embryo. The polar globules are dorsal, the macromeres are ventral. 
This does not, however, distinguish the anterior and posterior poles, and 
I know of no way in which they can with certainty be determined in 
Limax. The cells of the two ends are equal in size, generally, and when 
slight differences can be detected on careful measurement, it is impossible 
to follow these differences during the protean phases of cleavage that 
intervene between the two-cell stage and the appearance of the first 
mesoderm cell, marking the posterior pole. Inasmuch as the mesoderm 
cell (d*) comes from the left posterior quadrant, and is itself quite a 
large cell, while its sister cell (d’") does not seem to be much smaller 
than other members of its quartet, I have always placed the larger of the 
two touching quadrants of the basal quartet in the position /eft posterior, 
rather than right anterior. I have been compelled to orient arbitrarily 
in many cases, when no difference in size could be detected, choosing 
one of the two positions 180° apart. In choosing the larger cell I have 
not followed the type of Umbrella, where without doubt the mesoderm 
comes from the smaller of the two cells in contact at the ventral cross 
furrow. 


46 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


B. Discussion of Cleavage. 
SECOND GENERATION. First CLEAVAGE PLANE. Two CELLS. 
Plate I. Fig. 14. 


It should be noted that the phrase “ generation of cells ” is used in its 
strict genealogical meaning, and not in the sense in which Fol (75, ’76), 
Blochmann (’81), McMurrich (’86), and Heymons (93) have used the 
phrases “generation of micromeres ”’ and “ generation of cells.” The pro- 
cesses of maturation, fecundation, and the formation of the first cleavage 
furrow have been described in detail in Dr. Mark’s classic work upon 
Limax campestris. His published work was not carried beyond this 
stage, though he had continued his researches far into the later stages 
of development. The appearance of Fol’s work (80), which dealt. 
largely with the embryology of Limax, and the pressure of other duties, 
have interfered with the completion and publication of his studies. Dr. 
Mark has very kindly placed his numerous drawings and careful notes in 
my hands, and they have been of invaluable assistance to me in my work. 
I shall not attempt to add to his complete description of the formation 
of the first cleavage plane, but shall begin my work with the stage 
represented in Plate I. Fig. 14. This is a lateral view of an egg which 
has just completed the first division. Warneck (50) has stated that 
in Limax and Lymnzeus this plane is oblique to the axis of elongation, 
instead of transverse to it, and has distinctly said‘that this conclusion 
was not based upon a deceptive orientation of the egg. Fol (’75) has 
described a similar occurrence in Cymbulia. I have found no evidence 
that in the least confirms this statement of Warneck’s. At the stage 
shown in Figure 14 the two nuclei lie close to the approximated sur- 
faces of the blastomeres, at a level about midway between the animal and 
vegetative poles ; they are still quite small, and have only a very deli- 
cate membrane. Each has an elongated oval outline, with the long axis 
extending toward the astroceel of the cell in which it lies. Their 
position indicates that in the progress of the cleavage furrow toward the 
vegetative pole the nuclei (daughter segments) were in some way car- 
ried downward toward that pole. Mark (81) has described such a pro- 
cedure in the eggs of Limax campestris. There are a number of deeply 
staining granulations in the peripheral part of the cell adjacent to the 
nuclei, which probably constitute the remnant of the cell plate ; there 
is thus every indication of recent cell division. 

The astroccels appear as clear areas, almost as large as the nuclei, 
containing a few scattered deeply staining granules. These clear areas 


KOFOID: DEVELOPMENT OF LIMAX., 47 


have no limiting membrane ; they are, however, devoid of the granular 
structure of the surrounding protoplasm, and are the centres about 
which the radiations constituting the asters are arranged. 

The position of the astrocwls with reference to the nuclei is worthy of 
note. They are removed some distance from the nuclei toward the 
animal pole of the cells in which they lie. A comparison of this figure 
with that of a later stage shown in Figure 5 indicates that the astroccels 
are migrating toward a region where later the nuclei are found. It must 
seem therefore from the conditions in Figure 14 that the nuclei are pre- 
ceded in this migration by the astrocels. This recalls the shifting of 
male and female pronuclei attributed to the astroccels by Conklin (94) 
in Crepidula. 

In the living egg of this stage, when the cells have reached a perfectly 
spherical shape, each blastomere seems to be entirely independent of 
the other, and not the least trace of any contact or connecting proto- 
plasm can be detected between them. Each has a definite, unbroken 
contour, and in most cases there is an appreciable space between them, 
which shows no differentiation from the surrounding albumen. In the 
egg shown in Figure 14 the separation is not so great as it apparently is 
in the living egg. It is an interesting phenomenon, and raises the ques- 
tion as to the existence of any actual protoplasmic connection between 
the blastomeres in the stage following constriction. It is impossible to 
answer the question satisfactorily from observation of the living egg, 
for there is the possibility of the existence of a thin sheet of protoplasm 
which, on account of its transparency, thinness, and optical resemblance 
to the surrounding albumen, cannot be detected. The egg shown in 
Figure 14 was shelled by the process described in the preceding pages, 
and washed free from the albumen by normal salt solution, transferred 
in capillary tubes a number of times in the process of preparation, and, 
after mounting in balsam, was rolled over in various directions repeatedly 
without a separation of the two blastomeres. The two cells have each of 
them a definite and sharp outline at all planes of focusing, and even 
under high powers of the microscope no deeply stained granular bridge 
of protoplasm can be detected between them. It is only by very care- 
ful focusing that the rather vague, transparent, unstained connection 
between the cells can be seen. So far, then, as this preparation goes, it 
shows that there is a physical band of connection between the two 
blastomeres in this stage of greatest separation. The nature of this 
connection is problematical. It may be the Schleimschicht of Warneck 
(50), or it may be a continuation of the “ differentiated superficial 


48 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


portion of the yolk” noted by Mark (’81), there being no evidence of a 
well differentiated vitelline membrane. If the latter, then there is 
physical continuity in the living substance of the two blastomeres, and 
the appearance in the living egg is deceptive. Experimentation might 
possibly settle this interesting question. 

The two cells now lose their individual spherical contours, owing to 
their apposition and the mutual flattening of the two faces in contact. 
Thus the nearly spherical outline of the whole egg is re-established. See 
Figures 1-6, which form a series showing successive conditions of a single 
egg, and render a detailed description of the process unnecessary. 

The alternation of the rounded and flattened condition of cells during 
and subsequent to mitosis has been very generally observed in the stages 
of cleavage, but the explanation of the causes which underlie this 
phenomenon seems as remote as ever. Whitman (’78), in his discussion 
of the cleavage of Clepsine, concludes: “The cause of the separation 
and of the subsequent approach is undoubtedly the nucleus. . . . The 
proof that this is an electrical phenomenon is at present wanting, bnt the 
facts seem to point in this direction very strongly.” Our increased 
knowledge of the part that the cytoplasm plays in the process of cell 
division, especially the directive réle of the centrosomes, has suggested 
another influence to which we may turn for a solution, though the nature 
and exact operation of that influence is by no means definitely settled. 

This first cleavage plane divides the egg into equal or approximately 
equal cells. In some cases, by careful measurement, a slight difference 
in size could be detected ; in one case, for example, one of the cells 
measured 19 X 26 units of the ocular micrometer, and the other 20 x 27, 
when viewed from the animal pole. The theoretical consideration of the 
orientation of the early stages will be taken up later; suffice it for the 
present to say that the orientation adopted by Wilson (92), and later by 
Conklin 792) and Heymons (’93), will be employed in the present paper. 
The first cleavage plane, then, cuts the egg into an anterior half, A B, 
and a posterior half, CD, i. e. it is perpendicular to the antero-posterior 
axis of the egg. 

The discussion of the cleavage cavity will also be deferred till a later 
part of the paper is reached. 


THIRD GENERATION. SEcoND CLEAVAGE Furrow. Four CELLS. 
Plate I. Figs. 6-8; Plate Il. Fig. 15-18. 


The second furrow is formed, at the ordinary temperature of the labo- 
ratory, about two hours after the appearance of the first. Like the first, 


KOFOID: DEVELOPMENT OF LIMAX. 49 


it is preceded by an elongation of both cells in a direction at right angles 
to the plane of the division. Figure 15 (Plate II.) presents a view 
from the animal pole of a stage preparatory to this cleavage. The egg 
here represented is an exceptionally large one, being about 160 yw in its 
longest diameter. Each cell contains a spindle lying in its long diameter 
and nearer the animal pole. If the egg be viewed exactly from the 
animal pole, it is found that two of the asters —the rays of which are 
made more prominent in the figure than those of the remaining two — 
lie at a higher level than their mates. The same fact is brought out in 
a lateral view of this stage (Plate I. Fig. 7). Of the four asters the two 
having the same level lie in diagonally opposite quadrants of the egg. 
If we orient the egg so that the first plane of cleavage is transverse, no 
matter which pair of cells is placed anteriorly, and name the four asters 
A, L, C, D, in the accepted order, beginning at the left anterior quad- 
rant, we shall have the asters A and Cat the higher level, B and D at 
the lower. The slight difference in size between the two cells of this 
ege (Plate II. Fig. 15) has been previously noted. There is also a very 
slight difference in the stage of mitosis exhibited by the two cells, the 
larger being slightly more advanced than the smaller. A difference in 
the time of cleavage of the two cells of this stage has come under my 
observation in Limax a number of times. It is, however, not prevalent, 
and it is impossible to correlate it with any difference in the size of the 
two blastomeres. In Nereis, Umbrella, Cyclas, Unio, and many other 
forms, there is a well marked difference in size and a correlated difference 
in the time of division, the smaller cell being generally the first to divide. 

_ Figure 8 (Plate L.) represents the second furrow just before its com- 
pletion. The difference in level noted in the asters here finds its counter- 
part in the position of the partially formed blastomeres, the order of 
arrangement being the same as in Figure 15 (Plate II.). The planes of 
division are perpendicular to the axes of the spindle. They are there- 
fore not continuous, but both are oblique to the vertical axis and in 
opposite directions. The posterior plane (separating C and D) passes 
from above toward the vegetative pole and the right, the anterior (sepa- 
rating A and &) from above toward the vegetative pole and the left. 
Inasmuch as the two derivatives do not lie at the sume level, we may 
test the existence of the spiral; viewing the egg from the animal pole, 
and going from the lower derivative to the upper, we pass in a direction 
opposite to that in which the hands of a clock move; this oblique posi- 
tion of cognate cells may be referred to as a left spiral. It should be 


noted that this position of the cells is predetermined by the inclination 
VOL. XXVII.— NO. 2. 4 


“50 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


of the spindles, which exists before there is any constriction of the cyto- 
plasm. As in the case of the first cleavage plane, the constriction pro- 
gresses most rapidly from the animal pole. Toward the close of the 
process of constriction the daughter cells are united by only a bridge of 
protoplasm, which is nearer the vegetative pole. Figure 16 (Plate II.), 
from a preparation of a slightly older stage, shows a similar bridge of 
protoplasm, but it is much nearer the animal pole. 

The period of constriction is followed by a stage similar to that 
described for the two blastomeres, in which each of the four blastomeres 
assumes a spherical contour and stands out sharply and distinctly from 
its neighbors. This phase soon passes, and withiu half an hour the egg 
has assumed the condition of Figure 9 (Plate I.). This is the typical 
four-cell stage of the spiral type of cleavage, and therefore merits further 
description. (See diagram of this stage on page 52, Figure A.) The 
four cells, A, B, C, D, occupy the left anterior, right anterior, right 
posterior, and left posterior quadrants respectively. Each cell presents 
to the exterior a rounded, convex surface, and upon its inner side 
has three facets of contact, —the first and third with the cells of the 
adjacent quadrants, the second with the cell of the diagonally opposite 
quadrant. This last facet is triangular in shape, with its base at one 
pole and apex near the centre of the egg. The vertical axis of the egg 
lies in the planes of these central triangular facets. The bases of the 
central facets coincide with the well known cross furrows of the animal 
and vegetative poles of the egg (compare Plate II. Fig. 17). The cross 
furrow of the animal pole lies between the cells A and C, and extends 
from D to B, that of the vegetative pole lies between B and D, and 
extends from 4 to C. Thus by this mutual adaptation of the cells to 
one another, the spheroidal form of the egg as a whole is, in a degree, 
again restored, and here, as in the two-cell stage, persists during the 
period of “nuclear quiescence.” I have referred to the condition in 
Limax as “ typical.” I mean that the conditions are simple, and that 
the modifying influence due to the presence of a large amount of yolk, 
and its equal or unequal distribution among the four blastomeres, is 
absent. 

A comparison of the conditions presented here (Plate I. Fig. 9, Plate II. 
Fig. 17, and Fig. A, p. 52) with the same stage in other animals shows 
how profound the modifications are. In Limax the dorsal and ventral 
cross furrows are approximately equal in length, and as seen from the 
animal pole lie nearly at right angles to each other. In Nereis (Wilson 
*92) the dorsal furrow is largely obliterated, the four blastomeres almost 


KOFOID: DEVELOPMENT OF LIMAX. 51 


meeting in a point at the animal pole, while the ventral cross furrow is 
correspondingly longer. In Umbrella (Heymons ’93) the dorsal and 
ventral furrows of this stage are parallel, i. e. are formed between the same 
cells B and D, the cells A and C being considerably separated. This is 
undoubtedly due to the presence of a large amount of yolk in the four 
blastomeres. Likewise in Planorbis (Rabl ’79) and Neritina (Blochmann 
’81) we find the dorsal and ventral furrows of this stage similar to those 
of Umbrella, rather than Limax. These cross furrows are an invaluable 
aid in the determination of the axes of the later stages; the question of 
their relations and constancy will be discussed later, 


FourtH GENERATION. THIRD CLEAVAGE. E1Gut CELLs. 
Plate I. Figs. 8-13; Plate II. Figs. 17-19; Plate II. Figs. 20, 21. 


About two hours and a half intervene between the beginning of the 
four-cell stage and that of the eight-cell stage. The third cleavage is 
accomplished by the division of the quartet of the third generation, A, B, 
C, D, into two superposed quartets (cf. Figure B, p. 52), A*!— D*, 
and a4?—d**, The series of stages shown in Figures 8-13 (Plate I.) 
represents the egg during this process. Figures 17 and 18 (Plate II.) 
give apical and lateral views respectively of an egg with the spindles of 
this generation. It will be noticed (Fig. 17) that the spindles in no 
case stand vertically, but that they are inclined toward the right (right 
and left being used as resident in the egg; see my earlier paper, 794, 
p. 180). The division of the chromatin elements in the spindles has 
just begun, and there is no trace of a constriction of the cytoplasm. A 
comparison of Figures 17 and 19 shows that the degree of obliquity of 
the spindles has increased during the interval between the two stages. 
Figure 19 represents a stage in which the constriction of the upper from 
the lower quartet, i. e. of the micromeres from the macromeres, has just 
been completed. The obliquity increases during the division, so that at 
its close the cells of the upper quartet lie in the furrows between the 
cells of the lower quartet. Thus it will be seen that this apparent 
shifting of the upper quartet upon the lower, known as the “spiral,” 
takes place in large measure during the division of the cells. It will 
also be noted that the plane of division is at right angles to the axis 
of the spindle. 

If we view the egg from the animal pole and pass from the lower 
derivative of a pair to the upper, we move in the direction of the hands 
of a clock, and therefore the division of this, the fourth generation, takes 


52 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


place in a right spiral, whereas that of the third presented the character 
of a left spiral. Applying the system of nomenclature which I have 
adopted to the derivatives of the third generation, we find that cells of 
the lower quartet will be designated by the exponent 4.1, and the upper 
by 4.2. It will be convenient in the further discussion of quartets to 
refer to them simply by their exponents, without reference to the indi- 
vidual cells of which they are composed. It will be seen from Figures 
17 and 19 (Plate II.) that the dorsal and ventral cross furrows at the 
close of this stave do not lie at right angles to each other, as they did at 
the end of the four-cell stage, but that they cross each other at an angle 
as much less than 90° as is represented by the shifting of the cells to 
produce the spiral, i. e. they now cross at about 45°, as seen in the 
accompanying diagrams (Figures A and B). 


Ficure A. Ficure B. 


Figure A is a diagrammatic representation of the four-cell stage of 
Limax as seen from the animal pole, showing dorsal and ventral cross 
furrows. Figure B is the same of the eight-cell stage. 

This condition is not quite realized in Figure 21 (Plate III.), owing to 
the near proximity of the succeeding division, which restores the cross 
furrows approximately to the conditions of the four-cell stage. Thus, in 
the typical eight-cell stage of Limax the cross furrows correspond to 
those of the same stage of Nereis (Wilson 792, Plate XIV. Fig. 11). 
In Umbrella likewise (Heymons ’93, Taf. XIV. Fig. 4) the dorsal and 
ventral furrows are oblique to each other, crossing at about 45°, but 
differing in this important respect from the furrows of Limax and Nereis, 
that they are in this case formed by the juxtaposition of the cells of 
quadrants B and D at both poles, whereas in Limax and Nereis the 
ventral furrow only is formed by cells of these quadrants, the dorsal fur- 
row being formed by a*? and c*”, as is shown in Figure B. The furrows 


KOFOID: DEVELOPMENT OF LIMAX, 53 


in Neritina (Blochmann ’81) are similar to those of Umbrella. The con- 
ditions in the eight-cell stage of Planorbis (Rabl ’79) are complicated by 
the fact that this pulmonate probably has reversed cleavage (cf. Rabl’79, 
Taf. XXXII. Figs. 7, 9), and that therefore the mesoderm arises by a 
right spiral instead of a left one, as in the case of the unreversed or 
normal type of cleavage. Orienting the Planorbis egg for the purpose 
of comparison after the method employed by Wilson (’92) for Nereis, 
we have the first mesoderm cell arising from quadrant C’ instead of 
D; and in the four-cell and later stages the ventral cross furrow lies 
between A and C' instead of between B and D (cf. Rabl ’79, Taf. 
XXXII, Figs. 7, 8B, 11B). The differences between Limax and 
Planorbis will be best shown by a comparison of Figures A and B with 
the corresponding stages of Planorbis given in the diagrams below. 


Ficure C. Fiaure D. 


See 
EE) Cee 


Figure C is a diagrammatic representation of the four-cell stage of 
Planorbis, showing dorsal and ventral cross furrows parallel. Combined 
from Rabl’s ’79) Taf. XXXII. Figs. 7-12. Figure D, the same of the 
eight-cell stage. 

It will be seen that in Planorbis, as in Umbrella and Neritina, — all 
of them forms with considerable yolk, — the dorsal and ventral furrows 
are both formed by the contact of cells of the same quadrants. On the 
other hand, in Nereis and Limax the furrows of the two poles are 
formed by the contact of cells of different quadrants. In Nereis, how- 
ever, the dorsal furrow is comparatively shorter than in Limax, and in 
Umbrella it is of still less extent. 

I have observed no difference in the time of cleavage of the different 
quadrants. The nuclear conditions in Figure 19 (Plate II.) indicate 
that the division is very slightly more advanced in the posterior half of 


54 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


the egg, but there seems to be no acceleration of the division in the 
mesoderm-producing quadrant J over the quadrant C in this egg. 

The division, as has been before stated, is in individual planes oblique 
to each other, and not in a common equatorial plane such as occurs in 
radial division. In Limax the cells of the two quartets 4.1 and 4.2 are 
of unequal size, the inequality being almost as great as in Nereis; this 
will be seen on a comparison of my Figure 19 with Wilson’s (’92) Plate 
XIII. Fig. 10. We may therefore distinguish the components of the 
larger quartet as macromeres, and the smaller as micromeres of the first 
set. This difference in size, so marked at this period, persists in Nereis 
to a very late stage of development, but in Limax it is practically oblit- 
erated at the division leading to the next generation. Hence it is that 
a system of nomenclature based on these distinctions loses its significance 
when applied to the approximately equal cleavage of Limax. 


FirtH GENERATION. SIXTEEN CELLS. 


With the formation of the two quartets of the fourth generation it has 
become no longer possible to designate a single cleavage furrow as pro- 
ducing the next generation. I shall therefore discuss the cleavage, from 
this time on, from the standpoint of the successive cleavage of quartets. 


Division of Quartet 4.1, forming 5.1 and 5.2. 
Plate III. Figs. 20, 21. 


The basal and larger quartet of the eight-cell stage is seen in a 
mitotic condition in Plate III. Figs. 20 and 21. Here, asin the previous 
division, the spindles are not vertical, but much inclined ; this time, how- 
ever, the upper asters of each spindle lie to the /eft,’ and not to the right, 
of the lower ones (Fig. 20). The division of the chromatin elements 
has already taken place in the spindles of this quartet (best seen in 
Fig. 21), and the conditions of the completed mitosis can readily be 
inferred from the figures. If we view the egg from the animal pole, and 
pass from the lower derivative to the upper, we move in a direction 
opposite to that of the hands of a clock, i. e. this division takes place in 
a left spiral. The division of this quartet (4.1) is almost equal (Plate 
Ill. Fig. 22), the basal derivatives (5.1) being but slightly larger than 
the upper ones (5.2). In this respect Limax differs from all the yolk- 
laden forms, — Neritina, Planorbis, Umbrella, and Nereis, — where the 


1 Cf. Kofoid ’94, p. 180, for explanation of the use of right and left. 


KOFOID: DEVELOPMENT OF LIMAX. 55 


cells of the basal quartet (5.1) retain their preponderance, and may still 
be designated as macromeres after this division. 

The cell d*? (somatoblast of Wilson) is not appreciably larger than 
the other members of the quartet to which it belongs. 


Division of Quartet 4.2, forming 5.3 and 5.4. 
Plate III. Figs. 20-23; Plate IV. Figs. 28, 29. 


The same stages which show spindles in the lower quartet also exhibit 
them in the upper and smaller quartet. The mitosis is not however so 
far advanced as in the lower quartet. The nuclear membrane can still 
(Figs. 20, 21) be traced, though the asters are present, and the axis of 
the spindle can therefore be determined. This, as in the lower quartet, 
is inclined ; however, it is more nearly parallel to the equator than to 
the vertical axis. The inclination is in the same general direction as that. 
of the spindles of the lower quartet, and the conditions of the completed 
division can be inferred as readily from the figures. Viewing the egg 
from the animal pole and passing from the lower derivative or aster to 
the upper, we move in a direction opposite to that of the hands ofa 
clock, and this spiral, like that of the other quartet of this generation, 
is therefore a left one. This division, like that of the basal quartet, 
results in cells of almost equal size (Plate IV. Figs, 28, 29), the upper 
derivatives in this case (5.4) being, however, slightly larger than the 
lower (5.3). 

The conditions in Figures 20, 21, show that the sixteen-cell stage will 
in this case follow immediately upon the eight-cell, without the inter- 
vention of a well marked twelve-cell stage. There is, however, so much 
variation in the rate of cleavage in Limax, that it might be expected that 
a twelve-cell stage would occasionally make its appearance. We have 
but to increase the difference between the mitotic conditions of the cells 
of the two quartets of Figures 20 and 21 to produce. such a stage. 
Warneck (’50) figures in Tafel V. Fig. 46, a twelve-cell stage of Limax 
agrestis, and one egg in this stage has come under my own observation. 
This stage occurs regularly in forms with abundant yolk, as Planorbis, 
Umbrella, etc., but Nereis, like Limax, passes directly from the eight- to 
the sixteen-cell stage. The completed sixteen-cell stage is shown in 
Figure 22, Plate III., in which the genetic relations of the cells are still 
indicated by the approximated nuclei. 

With the completion of the sixteen-cell stage and the fifth generation, 
the dorsal and ventral cross furrows are restored to the conditions of 


56 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


the four-cell stage, i. e. they cross each other at approximately right 
angles. A similar restoration to the conditions of the four-cell stage 
occurs in Nereis, also in Umbrella at the twelve-cell stage, and probably 
in Neritina. 

In Planorbis, however, according to Rabl’s (79) interpretation, the 
cross furrow of the animal pole is not restored to the position of the four- 
cell stage, but is turned 90° from it (see his Taf. XXXII. Figs. 10 A, 
11 A). To accomplish this it is necessary for each of the cells of the 
apical quartet (5.4) to be sbifted 90° to the left, and thus completely 
out of their own quadrants over upon the adjoining quadrants. It seems 
very probable that Rabl is in error in this matter, and that in Planorbis, 
as in the other forms, the division of this generation results in the 
restoration of the cross furrows to the conditious of the four-cell stage. 

An examination of the sixteen-cell stage (cf. Figs. 21, 22) shows that 
the cells of the apical quartet (5.4) lie in the same meridian as those of 
the same quadrant in the basal quartet, i. e. a°* lies directly over a*', 
6*4 over 6°}, etc. ; a*®, 6°", etc. lie to the left of the meridian of the quad- 
rant to which they belong, and a®*, °°, etc. to the right. Thus of the 
four granddaughter cells of the original blastomere occupying the quad- 
rant, two only occupy the meridian corresponding to the middle of the 
quadrant, the other two being placed laterally to it, one upon either side. 
Similar conditions obtain in the corresponding stage of Nereis. In Neri- 
tina, Planorbis, and Umbrella, the fact that the twelve-cell stage is 
succeeded by the twenty-four-cell stage obscures somewhat the typical 
arrangement, though it can still be traced. It will readily be seen that, 
when the disturbing elements of unequal and non-synchronous division 
are removed, this arrangement of the four granddaughter cells will hold 
good for the descendants of any blastomere in spiral cleavage, and that 
normal and reversed cleavage will differ only in the transposition of the 
lateral granddaughter cells; e. g. in the case under discussion a*? and 
a** would be in a case of reversed cleavage transposed. Owing, perhaps, 
to the unequal distribution of the yolk, this typical arrangement is not 
found in the sixteen-cell stage of Chiton as figured by Kowalevsky (’83) 
and Metcalf (’93), though it can be traced in the later stages. Metcalf 
says of this phenomenon, “ Hach cell then lies in the same meridian as 
its grandparent, — a fact shown more clearly in the cleavage of such eggs 
as those of Nereis and Crepidula.” It is at once evident that this is 
but a partial and misleading statement of the case, since it ignores the 
fact that there are four granddaughter cells of every blastomere. It has 
its explanation in the confusing custom adopted in previous systems of 


KOFOID: DEVELOPMENT OF LIMAX. on 


nomenclature of regarding the larger of two daughter cells as the mother, 
the smaller as the daughter. In reality both are daughter cells, and 
the mother cell has passed out of existence with their origin, a fact 
which a logical system of nomenclature of cell lineage must always 
recognize. 


S1xtH GENERATION. THIRTY-TWO CELLS. 


Following the formation of the sixteen cells of the previous generation 
there comes the usual rounded condition in which each blastomere asserts 
its individuality and diverges from its nearest of kin. This in turn is 
followed by the flattened condition, in which the spherical contour of 
the egg as a whole is restored. It is during this period that the spindles 
which begin the formation of the sixth generation first appear. As in 
the previons generation there was a lack of synchronism in the cleavage 
of the two quartets 4.1 and 4.2, as shown in the nuclear conditions of 
Figures 20 and 21 (Plate III.), so here there is a similar separation 
of the divisions of this generation into two mitotic periods, the first 
appearing in the largest cells of the embryo, viz. the two quartets at 
the vegetative pole, and resulting in the twenty-four-cell stage ; the 
second involving the animal hemisphere, and resulting in the thirty-two- 
cell stage, thus realizing in this stage Warneck’s (50) conclusion that 
cleavage progresses according to the age of the cleavage spheres. The 
first phase is separated from the second by a period in which all the 
nuclei are in a resting condition. This period lasts some hours, and 
hence it is that cleavage stages killed at random contain a large pro- 
portion of eggs in the twenty-four-cell stage. 


Division of Quartet 5.1, forming 6.1 and 6.2. 
Plate III. Figs. 23-25; Plate IV. Figs, 27-32; Plate V. Figs. 33-36. 


Figure 23 (Plate III.) shows a lateral view of an egg at the completion 
of the first mitotic phase. The quartet 5.1 has divided, forming 6.1 and 
6.2. Applying the test asin previous generations, it is readily seen that 
this is a right spiral. The remnants of the nuclear spindle and the 
asters leave no question as to the relationship of the cells in this egg. 
The upper derivative lies to the right of the lower one in every case. 
Reference to the quartets 6.1 and 6.2 in Figures 27-29, 31-34, 36, of 
four other eggs, shows the constancy of the direction of this spiral. The 
division in this quartet is about equal, and is synchronous in the four 
quadrants. 


BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


o. 


Division of Quartet 5.2, forming 6.3 and 6.4. 


Same Figures as for 5.1. 


Figure 23 shows the quartet at the close of the division which has 
resulted in the formation of the quartets 6.3 and 6.4. The nuclear con- 
ditions in like manner show that this division has also taken place in a 
right spiral. The remnants of the spindles are parallel to those of the 
quartet 5.1, and the upper derivative lies to the right of the lower one. 
These divisions are also approximately equal and synchronous. The 
arrangement of the cells of the vegetative pole is very regular. The ven- 
tral cross furrow remains approximately at right angles to the dorsal 
furrow. The two cells 0°! and d*! meet in this ventral furrow, and are 
hexagonal in outline, while the other two members of the quartet a*" 
and c*! are pentagonal. All four cells of the quartet 6.2 are hexagonal, 
as will be seen in Plate IV. Figs. 27-29, and 31. A comparison of the 
quartet 6.1 of Figure 27 with 5.4 of Figure 28 shows how little differ- 
ence there is in the size of the cells of the two poles in the superficial 
view. In optical section, however, a distinct difference can be detected 
in the size of the deeper lying parts of these cells. In Figure 30 (Plate 
IV.) is represented such a section taken from the egg in the position 
shown in Figure 29. The section passes through the vertical axis in 
the plane corresponding to that of the paper in Figure 29, thus cutting 
the ventral cross furrow at right angles, and passing through the quad- 
rants band d. It will be seen that one of the two cells of the vegeta- 
tive pole, labelled d*! in the drawing, is much larger than the other, 0°. 
This is the cell which at the next generation gives rise to the first 
mesoderm cell, d7*, or M. 

The generalization which Rabl made in his paper on Planorbis (’79), 
— “dass bei den Keimen mit reichlichem Nahrungsdotter von dem Zeit- 
punkte an, als Aequatorialfurchen auftreten, die Zellvermehrung in 
arithmetischer, bei den Keimen mit spiirlichem Nahrungsdotter dagegen 
zuerst in arithmetischer, sodann aber in geometrischer Progression 
erfolgt,’ — is not borne out by the cleavage in Limax, as the twelve-cell 
stage does not regularly occur. The cleavage in Limax runs, 


CS een a ae a Pe ee eee ae 
or, 4:>4M:M4+4:4M+0:@74%+@44 ©). 
This is fundamentally an arithmetical progression, a series whose common 


difference is four, the mathematical expression of the increase in the 
number of cells in the spiral type of cleavage. It varies, however, from 


KOFOID: DEVELOPMENT OF LIMAX. 59 


the strict arithmetical series in that a multiple of the common difference 
is sometimes added as a result of the approximated or even synchro- 
nous division of two or more quartets. Thus the series may in some of 
its parts, like the twelve- to twenty-four-cell period of Planorbis, take 
the form of a geometrical series; but this is not fundamental, and, as 
Warneck in 1850 expressed the law, ‘In jedem Stadium des Furchungs- 
processes entstehen nur vier Furchungskugeln, d. h. die Theilung geht 
nicht in einer geometrischen sondern einer arithmetischen Progression 
vor sich.” 

The general contour of the egg in the twenty-four-cell stages figured 
(Plate III. Figs. 23-25, Plate IV. Figs. 27-32, Plate IV. Figs. 33, 34) 
has been spherical, the transverse and vertical diameters being approx- 
imately the same. In the period leading up to this stage, however, 
eggs are found showing a considerable flattening in the dorso-ventral 
direction, i. e. a shortening of the vertical axis. In one egg showing 
the flattening, the dorso-ventral diameter was 70 w and the transverse 
120 uw. This change may be dependent upon an elimination of such a 
cleavage cavity as is shown in Plate V. Fig. 34. A similar flattening is 
often found in those eggs in which the divisions leading to the forty- 
cell stage are taking place. These divisions will now be discussed. 
The order of their discussion does not, however, indicate their chrono- 
logical succession. 


Division of Quartet 5.3, forming 6.5 and 6.6. 
Plate VI. Figs. 37, 39, 41. 

The division of this quartet does not take place, in some instances at 
least, until after the forty-cell stage ; i. e. it is accompanied by divisions 
of the succeeding generation in other quartets. See Figures 39 and 41. 
In Figure 39 the cells a>’ and d** have divided, and 6** is in a mitotic 
state, but c’* is as yet undivided. In 6°* the end plates have been formed, 
but the cytoplasm is not yet constricted. The axis of the spindle lies 
parallel to the plane of the equator. There is every indication that the 
division is very nearly meridional. Figure 39 has forty-two cells; in 
Figure 41 there are forty-five cells; here, however, it is cells a’ and 
d>* that have but recently divided, the other cells of the quartet having 
evidently been divided for some time. Thus there is no constancy as 
to the sequence in which the cells of this quartet divide. In Figure 
41 (Plate VI.) this division still shows some slight traces of a right 
spiral. Figure 37 (Plate-VI.), a forty-cell stage, is described in my notes 
as containing the cells a®°-d*®, a®>-d**, but owing to the rotundity 


60 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


and opacity of this egg the details in the periphery, i. e. the region of 
the cells in question, are very much obscured, and I consider this deter- | 
mination questionable. 

The comparison of the division of this quartet with the corresponding 
one in Nereis is very interesting. In Nereis, as in Limax, the division 
is nearly meridional, and with traces of a right spiral. In this instance 
it takes place at the twenty-nine-cell stage, and the products form the 
prototroch. In Umbrella it does not take place till the sixty-five-cell 
stage, and here also exhibits an obscure right spiral. (See Tables of 
Cleavage, pp. 66, 74, and 75.) 


Division of Quartet 5.4, forming 6.7 and 6.8. 
Plate V. Figs. 35, 36; Plate VI. Figs. 37, 39, 41. 


This is one of the first divisions to follow the twenty-four-cell stage. 
Figures 35 and 36 (Plate V.) show it in progress; Figures 37, 39, and 41 
after completion. It takes place in a very evident right spiral, the upper 
aster and derivative lying to the right in every case. The division is 
approximately equal, but is not synchronous in the different quadrants, 
as is shown in Figures 35 and 36. In Figure 35 all the cells of this 
quartet have divided except a*4; the quadrant c, judging from the size 
and position of the daughter nuclei, has evidently led in the division. 
In Figure 36, 6°4 is the only one which has divided, resulting in J°7 and 
b°8, the other cells containing spindles. It is evident from these two cases 
that it is impossible to predicate any regular sequence in the successive 
divisions of the quadrants of this quartet. 

This completes the discussion of the cleavages of this generation. It 
will be noted that all of the divisions clearly take place in a right spiral, 
with the exception of that of 5.3, and that this, though predominantly 
meridional, still shows traces of a right spiral. 


SEVENTH GENERATION. SIXTY-FOUR CELLS. 
As was stated in my earlier paper (94, p. 188), the divisions of this 
generation begin before those of the sixth are completed. 
Division of Quartet 6.1, forming 7.1 and 7.2. 
Plate VI, Figs. 38, 40, 42. 


The division of this quartet is the point of greatest interest in the 
cleavage, as it results in a differentiation of the germ layers, or at least 
in a separation of the primary mesoderm from the ect-entoderm. 


KOFOID: DEVELOPMENT OF LIMAX. 61 


In spite of the examination of a large number of eggs, and the repeated 
killing of those whose age and approximate stage were known, I have not 
been able as yet to obtain an egg showing the spindles resulting in this 
division. Figures 38, 40, and 42 (Plate VI.) all represent stages sub- 
sequent to the formation of J/, and the other members of the quartet 
7.2. There is some evidence, however, as to the character of the divis- 
ion. Figure 38 is a view of the vegetative pole of an egg of forty cells. 
The four central cells have seven peripheral neighbors. Deeper focusing 
reveals the presence of a large nucleus, lying within a definite cell 
boundary. This is quite below the level of the nuclei of the vegetative 
quartet. Its nucleus lies below and slightly nearer the median plane 
than that of d’4. The superficial extent of this deeper lying cell is 
limited to a narrow strip adjoining the cell with which it has arisen, 
i.e. it is peripheral to d™'. The other members of the quartet 7.2 are 
present, and when the test for the spiral is applied it is evident that 
this is a left spiral, though the amount of the shifting is evidently not very 
great. It is quite plain that in this case the cell d*’, which gives rise to 
the mesoderm, comes from d*! at the time of its division into d™ and 
d’’. As in previous stages, the quartets d and 6} are in contact at the 
ventral cross furrow. 

Figure 40 (Plate VI.) represents a forty-two-cell stage which has 
recently been the scene of a number of divisions. Here, as in the egg 
just discussed, the cell d’* is very large, and is crowded in toward the 
centre of the egg, lying below d™ and slightly nearer the median plane. 

‘It maintains a small crescentic connection with the exterior, between 
d™ and d®**. The other members of the quartet: 7.2 are present, and 
show about the same nuclear conditions that d™* (= MW) does. They 
are therefore of about the same age. ‘The divisions of the cells of the 
quartet 6.1 in this egg have evidently been very nearly equatorial, and 
unless there should be at a subsequent period some shifting of the 
mesoderm to a position nearer to the median plane, —as adopted in this 
paper, — it would be necessary to orient this egg as Rabl and Blochmann 
have oriented Planorbis and Neritina. Judging from the nuclear condi- 
tions the division has taken place quite recently. Eggs of later stages 
show that the mesoderm is generally placed bilaterally with reference 
to the cross furrows and the prevailing quadrangular form of the egg 
of those stages. The division of this quartet is in all cases unequal, the 
smaller cells lying at the vegetative pole. 

The conditions of the egg shown in Figure 42 (Plate VI.) were for a 
long time very puzzling to me. It contains forty-five cells, which 


62 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


readily group themselves into quartets. A median deep-lying mesoderm 
cell is present, as in the egg last described, but the relations of the cells of 
the quartet of the vegetative pole to this cell and to one another are differ- 
ent from those of the other eggs, in the following respect : the cells which 
meet in the ventral cross furrow are a” and c”!, instead of d™ and 671, as 
in the other two eggs figured. Repeated trials failed to give any other so- 
lution which would accord with the conditions in the other parts of the egg. 
The juxtaposition of the quadrants a and ¢ occurs normally in the reversed 
type of cleavage (see Figs. C and D, p. 53), and the possibility is at once 
suggested that this egg may have had reversed rather than unreversed or 
formal cleavage. Other parts of the egg, however, furnish no corroborative 
evidence, and the suggestion must be dismissed. I believe, then, that 
owing to some cause, mechanical or other, a change in the normal rela- 
tions of the cells of the quartet to one another has been brought about. 


This has naturally raised the question as to the constancy of the cross. 


furrows, upon which the orientation of the egg so largely depends. 
This case in Limax is not an isolated one, for in Nereis, where, as has 
already been pointed out, the dorsal furrow is formed in the early stages 
of cleavage by the apposition of the quadrants @ and c, we find this 
normal arrangement disturbed in one instance, the furrows being formed 
by the quadrants 6 and d (Wilson 792, Plate XIV. Fig. 19, p. 390). In 
the later stages, i. e. after the cells of the seventh generation appear at 
the animal pole, the dorsal cross furrow is’ normally formed by the 
apposition of 6 and d, but in one case (Wilson /. ¢., Plate XVI. Fig. 35) 
we find this arrangement disturbed, the cross furrow being formed by a 
and c. This disturbance is also accompanied by the mitotic conditions 
of neighboring cells. 

Another case occurs in Neritina (Blochmann ’81, Taf. VIT. Figs. 51, 
53, 56), in which two eggs —one a thirty-six-, the other a forty-cell 
stage — present cross furrows formed by the apposition of different pairs 
of quadrants. There is not here, as in Nereis, an intervening mitosis to 
explain the disturbance of the customary order. 

In Lang’s (’85) Taf. 34, Figs. 14, 15, we find a similar transposition 
from the usual arrangement of the apical quartet, accompanied in this 
case by mitosis in adjoining cells. In view of these cases it seems not 
improbable that there has been in this Limax egg a disarrangement 
of the normal condition at the vegetative pole, as a result perhaps of the 
recent divisions at that pole, the collapse of the cleavage cavity, or some 
other mechanical disturbance. 

It seems almost certain that the primary mesoderm cell, d’? (17), is 


pin Ree ORT 4 


*KOFOID: DEVELOPMENT OF LIMAX. 63 


formed synchronously with the other members of the quartet to which 
it belongs. In this respect Limax stands in sharp contrast to Nereis, 
where the primary mesoderm cell originates at the thirty-eight-cell stage, 
but the cleavage of the remaining cells of the quartet is long delayed. 
Also in Umbrella there is a corresponding lack of synchronism, for the 
division of this quartet commences with the formation of d’* at the 
twenty-five-cell stage, but is not completed until the forty-seven-cell 
stage is reached. Likewise in Planorbis the formation of the primary 
mesoblast antedates the cleavage of the other cells of the same quartet. 


Division of Quartet 6.2, forming 7.3 and 7.4. 


Plate IV. Figs. 31, 32; Plate V. Fig. 353 Plate VI. Figs. 38, 40. 


The spindles resulting in this division are among the first to appear 
in the twenty-four-cell stage. Figures 31 and 32 (Plate IV.) show 
spindles in all of the cells of this quartet except a®*, and in this the 
stages preparatory to the formation of the spindle are seen (Fig. 31). 
The nucleus is very large; the chromatic granules are distributed in a 
network, and the nuclear membrane is still intact. At diametrically 
opposite sides of the nucleus, in the long axis of the cell, and closely 
applied to the nuclear membrane, there are two large, clear spherical 
spaces in the cytoplasm, bounded by a granular zone. These structures 
are probably the astroceels of Fol (91). The surface of the nucleus pre- 
sents on one side a peculiar constriction, or crease, running between the 
two astroccels, as though they were connected by a strand of substance 
(central spindle) which was compressing the thin nuclear membrane. 

The spindles in this quartet, as shown in Figures 31 and 32 (Plate 
IV.) are almost free from any inclination indicative of a spiral arrange- 
ment. The one in 6°? shows traces of a right spiral, but there is indica- 
tion from the position of the spindles that the division will be equatorial 
rather than oblique ; such indeed is the character of the division, as is 
shown in c’*, c’4, Figure 35 (Plate V.). The order of nuclear advance- 
ment in this quartet as shown in Figure 32 (Plate IV.), is }, d, c, a, but 
in Figure 35 (another egg) the cell ¢ has been the first to divide, whereas 
in Figure 38 (Plate VI.), a forty-cell stage, the cell d*° is just dividing, it 
being the last of its quartet to undergo the process. This seems to show 
either that the cleavage in this quartet progresses very slowly, or, what 
is more probable, that there is considerable variation in the sequence in 
which its components divide. In the case of Figure 38 (Plate VI.) 
there is strong indication of a left spiral ; so also a slight indication of 


64 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


the same in Figure 40, a7’, a’*. In all the other figures the division 
seems to be equatorial. A comparison with Nereis reveals in this 
instance the same marked agreement noted for the meridional cleavage _ 
of the quartet 5.3. In Umbrella this division takes place at the twenty- — 
nine-cell stage, and is also equatorial. 


Division of Quartet 6.3, forming 7.5 and 7.6. 
Plate VI. Figs. 41, 42. 


This division is in progress in the quadrant ¢ in Figures 41, 42, and 
the other members of the quartet are also approaching mitosis. There 
is a faint trace of a left spiral to be detected in the position of c’* and 
c’® of Figure 41, but the division is predominantly equatorial. 


Division of Quartet 6.4, forming 7.7 and 7.8. 
Plate VI. Figs. 39, 40, 41, 42. 


In the two eggs figured the spiral is clearly shown by the relative 
position of the nuclei to be a left spiral. Thus all the spirals of this 
seventh generation, wherever they have been traced, have been left 
spirals. 

With this forty-five-cell stage my detailed account of the cleavage 
closes. I have not been able to decipher satisfactorily the conditions in 
the eggs of the next stage, because during this stage a large number of 
cells divide, —in one instance as many as thirteen. Moreover, the 
rounded contours of the mitotic cells produce such changes in the surface 
of the egg as effectually to obscure all trace of its poles, and the absence 
’ whatever for 
orientation, makes any interpretation of these later stages at the best 
provisional, and very largely conjectural. Added to these difficulties is 
that produced by the vacuolation which prevailed in a very large pro- 
portion of the eggs which I have examined. This distorts and obscures 
the relation of cells to such an extent as to make a determination of 
their lineage extremely difficult, if not impossible. 

As late, however, as the hundred-cell stage, when four mesoderm cells 
are present, it is possible on favorable eggs to work out a provisional — 
lineage, but I have not as yet succeeded in connecting this with the — 
forty-five-cell stage. 

Thus the outcome of my work as a study in cell lineage is 2 disappoint- 
ment, for I have not been able to trace a single blastomere to a definite © 
organ of the adult. At the stage of thirty-eight cells in Nereis, Wilson 


of polar globules, of macromeres, or of any “landmarks ’ 


KOFOID: DEVELOPMENT OF LIMAX. 65 


was able to assign a definite fate to each blastomere; but in Limax 
there is no trace, save in the early differentiation of the mesoderm, of 
that precocious development so marked in Nereis. This fact makes the 
identity of the cleavage of Limax with that of Nereis all the more won- 
derful and difficult to explain. 

I insert here (p. 66) a table of the cleavage of Limax, so far as I have 
followed it, which epitomizes the foregoing discussion of the alternation 
of spirals in successive generations of cells. The spirals, wherever they 
occur, conform to the law of alternation as defined in my former paper 


(94, p. 189). 


C. Literature on Spiral Cleavage. 


The conformity of other animals to the law of spiral cleavage has in 
all cases been obscured by the systems of nomenclature employed. 
Since no one of my predecessors has formulated this supposed law, it of 
course has not been tested on any of the forms whose cleavage has been 
worked out. It has seemed desirable, therefore, to go over the available 
literature and point out those cases which agree, and those which seem 
to disagree with my proposition. 

In order that the subject may be treated in as brief a form as possible 
the discussion of each case is accompanied by a tabulated presentation 
of the cleavage, in which the author’s designation of cells and spirals is 
joined in parallel columns with the designation which my system would 
impose. 

In my former paper (94, pp. 192-196) the conformity of the cleavage 
of Neritina, as described by Blochmann (’81), to the alternation of spirals 
was discussed, and the cleavage tabulated. In what follows I have 
discussed all other cases which seemed worthy of consideration in this 
connection. 

Fol states (75, p. 117) that Clio likewise has the same regular cleav- 
age as Cavolina, and his few figures of the early stages of this form 
suggest that the cleavage is of the normal type. Cymbulia also seems 
to conform to this type. 

The cleavage of the Heteropods, which he (’76) states is identical 
with that of the Pteropods, is, according to his figures, of two types: 
Firoloides (Plate I. Figs. 1-3) presenting the normal type, Pterotrachea 
(Plate IV. Figs. 5, 6) the reversed type, if his labelling, indicating the 
lineage, 1s correct. There is evidence, however, that some of the divis- 
ions belong to the normal type (Plate IV. Fig. 9). . 


VOL. XXVIT. — NO. 2. 5 


66 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


CLEAVAGE OF LIMAX. 


Generation. |Number of Cells, Designation of Cells. Spirals. 
75%) 
6.4 4 Left. 
40 a << av | 
7.60 
48 a®:3 << Pi Left, — cleavage nearly 
a’) equatorial. 
VIL. _ 
x ae 
28 ae <A ee ; Left, — cleavage nearly 
equatorial. 
7.2 
rg hho 
36 abl os ) Left. 
a’) 
32 qo-4 a a Right. 
a -f 
6.6 
44 a®3 we Right, — cleavage nearly 
~ Gs meridional. 
VL. - 
24 q>:2 < me a Right. 
Gs) 
6.2 
5 are ae eae d Right. 
20 a <a ae) 5 8 


5.4 
16 as a oe Left 


ip 12 MAS EG Left. 
IV. 8 es yeh Right. 
III. 4 ALB aD: Left. 
I 9 AB, CD 


KOFOID: DEVELOPMENT OF LIMAX. 67 


As stated in my earlier paper (94, p. 191), there are some indica- 
tions in Rabl’s work that Planorbis presents a case of reversed cleavage, 
i. e. the cleavage is such that the spirals of the even generations are left, 
and those of the odd right. Fol’s (’80) figures of the eight- and six- 
teen-cell stages of this Pulmonate (Plate IX.—X. Figs. 1, 2) indicate 
that the spiral of the fourth generation is a left one, and that of the 
fifth a right one. My own sketches of Planorbis, made from living 


CLEAVAGE OF CAVOLINA. 
Fol (’75). 


Fou’s NOMENCLATURE, REVISED NOMENCLATURE. 


Gener- |Number 
ation. of 


Spirals.1 S. ; Spirals. 


Right. 


Right. 


Left. 
La TTT VN ALE. 4 . | Division oblique, 


cells in one plane. 


eggs in the spring of 1892, show the same direction in these spirals. 
So far as can be judged from these facts, the alternation of spirals 
holds in this case, which is probably one of reversed cleavage. 

If the reversed spirals persist until the formation of the mesoderm, this 
germ layer would then come from the right posterior macromere, instead 

1 Fol does not name or discuss spirals) The nomenclature in this column is 
deduced entirely from his lettering of the cells. After the third generation the 
divisions of only one quadrant (Z) are followed. In this, as in the following tables, 


capital letters indicate macromeres, and the lineage of only a single quadrant (a of 
the revised nomenclature) is given. 


68 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


of the left posterior one, i. e. if we adopt Wilson’s orientation instead of 
Rabl’s. In the table given above it will be noticed that the mesoderm 
cell M/ is placed as a member of the basal quartet of the sixth generation. - 
It should be remembered, however, that Rabl in a later paper (80) 
revised this earlier opinion as to the origin of the mesoderm cell, deriving 


CLEAVAGE OF PLANORBIS. 
Rabl (’79). 


RaBi’s NOMENCLATURE, REVISED NOMENCLATURE. 


Spirals. 


JT OPN Ue 
ee ee, 


By, Ly, Lis a || AS ch) mete 
EJ, BJ, BJ, M | 
i 
5 


Left ? 


E, 


5 


EU. a a ME 


ENS Ben Bi, 
EJ, EJ, EJ, ME 


EJ, EJ, EJ, ME 


it this time by division from the cell Jf of the above table. According 
to this the mesoderm cell in Planorbis, as in many other forms, belongs 
to the quartet 7.2. It seems to me that the orientation of the stages of 
Planorbis previous to the formation of the mesoderm is still an open 
question. As I have stated in the discussion of Limax, the orientation 


' 
‘ 


a eee 


es 


KOFOID: DEVELOPMENT OF LIMAX. 69 


of the early stages depends on the estimated amount of shifting of the 
mesoderm cell upon the basal quartet a71— d". There is evidence in 
Rabl’s figures (80, Taf. XXIX. Figg. 2, 3, 4) that there is at least some 
shifting, but whether it is sufficient to justify an orientation similar to 
that adopted by Wilson for Nereis cannot be decided from the evidence 
at hand. It is however sufficient to raise the question as to the cor- 
rectness of Rabl’s orientation. 

In Planorbis the asymmetry of the adult is sinistral ; so also in Physa 
heterostropha, whose segmentation likewise shows the same phenomenon 
of reversed cleavage, as I can affirm from my own observations on the 
living egg.t In my former paper (’94, p. 191) I called attention to the 
fact that Haddon (82, Plate XXXI. Fig. 6) figures a four- to eight-cell 
stage of Janthina which apparently presents a left spiral, i.e. the reverse 
of that found in a corresponding stage of Limax and other dextral forms. 
But according to Fischer (’80-87, p. 775) Janthina is a dextral form, 
These cases of Planorbis, Physa, and Janthina at once raise a most inter- 
esting question as to the relation that may exist between the two forms of 
cleavage, normal and reversed, and the dextral or sinistral asymmetry of 
the adult. The occurrence of reversed cleavage in these two sinistral 
forms is suggestive of a causal relation between the conditions of the em- 
bryo and the adult, but the existence of this one observation of Haddoun’s 
is sufficient to throw much doubt on that conclusion. There remains the 
possibility that on renewed examination the cleavage of Janthina may 
prove to be normal, and that this case of Haddon’s may be referred to a 
chance sinistral form. In answer to an inquiry of mine, Dr. J. I. Peck 
of Williams College has kindly written, “I have never seen a sinistral 
Janthina; all of those which came under my observation (’93) were 
dextral: all of those in our collection here are dextral also, as were 
those I saw at the Museum at Yale. These were of four species, I think, 
and collected at different points of the Atlantic and Pacific, both Arctic 
and Tropical. I have, however, probably not seen enough material to 
expect a sinistral individual.” Of the two or three hundred shells of 
Janthina, representing several species, in the Museum collection, not one 
is sinistral. 

The data for the discussion of this interesting question are as yet too 
scanty to justify any generalizations. The examination of the cleavage 
in such genera as Fulgur and Achatinella, which contain both dextral 
and sinistral forms, as well as that of heterostrophic species, ought to be 
decisive upon this point, where we have as yet so little light. 


1 Consult also the Addendum, page 111. 


70 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


CLEAVAGE OF APLYSIA. 
Blochmann (783). , = 


BLOCHMANN’S NOMENCLATURE. 


Meridional, 
a‘? | slightly left. 


Left. 


In the above table the labelling of cells indicated in the second col- 
umn is that given by Blochmann to cells which seem to be homologous 
with those of quadrant A. 

The cleavage of Aplysia, so far as followed by Blochmann, resembles 
very closely that of Umbrella. If we interpret the cleavage as Heymons 
has that of Umbrella, we find that here also the posterior and smaller 
cell of the two-cell stage divides first; the mesoderm quadrant D 


KOFOID: DEVELOPMENT OF LIMAX. tL 


(labelled ¢ by Blochmann) also precedes in some instances in the later 
divisions. The yolk, however, is confined mainly to two of the macro- 
meres, A and JS, instead of being lodged principally in three, A, 2, and 
C, as it is in Umbrella. 

All attempts to reconcile the cleavage of Chiton, as described by 
Kowalevsky (83) and Metcalf (93), with the alternation of spirals 
shown in other forms of Molluscan cleavage are involved in serious 
difficulties. Unfortunately neither author figures nuclei or spindles, 
with the exception of one figure in each paper. The relationship of 
cells and the direction of the spiral is also explicitly stated in certain 
cases to be a matter of conjecture on their part. Therefore it does not 
seem profitable for me to add to their conjectures others of my own. 
It is however possible to force upon the cleavage of Chiton, as figured 
by these authors, an interpretation which causes it to accord with the 
spiral type of cleavage, but this interpretation meets a serious obstacle 
in the sixteen-cell stage, though it does not violate the relationships 
of cells in the few cases where these authors have indicated relation- 
ships by nuclear conditions (Kowalevsky, Plate I. Fig. 7, Metcalf, Plate 
RV. Fig. xvii.). 

I do not wish to commit myself to the view that Chiton conforms to 
that type, for, as Metcalf has suggested, “ most of the divisions are 
of the radial type.” The distribution of the yolk in the blastomeres is 
also suggestive of the radial type. There are, however in Kowalevsky’s 
figures (Plate I. Figs. 4-13) many suggestions of spiral cleavage. In 
Metcalf’s figures, on the other hand, “ individual variations in the shape 
of the blastomeres are not preserved. The figures show what may be 
called the typical condition.” Thus most of the evidence of spiral 
cleavage, if such exists, must be eliminated from his figures. 

The cleavage of Discoccelis conforms to the spiral type and the alter- 
nation of spirals, with the possible exception of the spiral of 7.15-7.16, 
to which reference was made in my former paper (’94, p. 196). It is 
necessary to relabel Figure 6 of Lang’s Tafel XXXV., in order to reconcile 
it with the apical views which he has given of the same stage, and also 
with the principle of alternation. 

The conformity of the cleavage of Nereis to the law of alternation of 
spirals is perfect, as is demonstrated by the application of the uniform 
system of naming spirals in the above table. Even in those cleavages 
designated as equatorial or meridional, traces of the spiral characteristic 
of the generation can often be detected. 

The Nereis table is marked by the abrupt termination of the spiral 


(2 


CLEAVAGE OF DISCOCGLIS. 
Lang (’85). 


BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


REVISED NOMENCLATURE. 


Lane’s NOMENCLATURE. 
Gener Number 
ation. eae 
Spirals, Cells. Cells. 
7.16 
ae 6.8 a 
Left. 1'> ae 44 a’ 
ate, > aey <i qils 
7.14 
ae. 6.7 a 
— ae = ae, 40 a rte ae 
WAUE 
am, > am 52 pee qis 
am 1 a qui 


| Spirals. 


Right, contra- 
dicts law of 
alternation. 


Left 2 
Left. 


Left. 


KOFOID: DEVELOPMENT OF LIMAX. 73 


CLEAVAGE OF NEREIS. 
Wilson (92). 


Witson’s NOMENCLATURE. 


RevisED NOMENCLATURE. 


G Number 
zener- of 
ation, Cells 
Spirals, Cells. Cells. Spirals. 
ais 1 6:8 a7-16 Left, c and d 
Left. A :. % as . < ails precede. 
1.2.2 7.14 
Bete ||, > al — | arc Left. 
1.1.2.2 12 
Horizontal. My ae 42-58 | a®6 ae Equatorial. 
Tef.12 7.10 
Vertical. [fyia > a 42-58 | a®5< fa Meridional. 
xX e VII. di8 
— 2 > x 37+ | dé-4 qui | Left, d precedes. 
Horizontal. Es ope = =|) aise <ae a Equatorial. 
Horizontal. i a3 ee << a Equatorial. 
M di2=M 
Left. D ie: D 38+ | Ds-1 < 71 | Left, D precedes. 
1 6 
Left. 2 al 20 a oe Right 
nes Velie2 6-6 Ri ht vh- 
: 1 2 5.3 ight, approach 
Mepacignal aoe > m VI ? = << a’ ing meridional. 
2.2 \ 6.4 
Left. ne a? 23-32 | a5-2 ae Right, d precedes. 
: a3 9 5.1 he Right, C and D 
Right. a > A 23-29 | A << ‘Ab recent 
Right. | % 1 i eee OO 
bist qual = a a ee qo-3 Left. 
2 V. 5.2 
Left. wi = A 16 et Mea Left, D precedes. 


Right, C and D 
precede. 


Left. 


74 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 
CLEAVAGE OF UMBRELLA. 
Heymons (’98). 
Heymons’s NOMENCLATURE. Gener. Number REVISED NOMENCLATURE. 
3 I ma 
Spirals. Cells ation. | Cells. Cells. | Spirals. 
ee) a3 ” 15 8.14 anes +3. 
an sg “= 2 << 9-21 Meridional. 
” ae “a 9.26 
Right. eee ; > Gee 79 | q8-13 ee Left. 
Die, D 17 a’-l4 Equatorial, 
ee a te hs << a8-13 | trace of right. 
uw 8.12 
—— a” a’ bp) | até ae 8.11 Equatorial. 
Left, a and b. Gee Right, a and b. 


Merid., c and d. 


Meridional. 


Like Nereis. 


Right. 
Left. 
Left. 
Left. 
Left. 
Left. 
Right. 
Right. 
Right. 
Right. 


Left. 


Right. 
Left. 


Merid., c and d. 


Meridional, 
a and b precede. 


Bilateral, 
trace of right. 


Left. 


Left. 


Left, > and 
c precede. 


Left, 6 and 
d precede. 


Equatorial, 
trace of left. 


Left, D precedes. 


LED) 
a ae me as6 
on > a ‘1 67 ai3 <i ane 
A” 5 8.2 
no PTE Baw 
‘ qi-16 
Not figured. 914-| ae <e qi-16 
, 7.14 
an > a’s 7Al ast =O nae 
2.1 
a z ai 
“1 > ay 33 a’-4 < qi 
a 
ee VII. es Es qi-6 
4/ as 
a’ e a 37 ae 
7.4 
ine ji Qe ee 
a; = a 29 << qi 
7.2 
As a 25-47 Ab-l A’ 1 
a = Al: 
, 6.8 
a a 44 | ait ao 67 
oe f * qb-6 
a's & Zo VI ore <— af.5 
ll o = 5 4 
a” = & 24 a? << q5.3 
a” : : qo-2 
Aeris 161) Abt “fea 
wv >a’ 20 ae Os 
a’; Vv qd3 
Za ; 5.2 
Lee 12 | AM Maa 
ii at2 
GE SiAs WAY, 2) ee | ee gan 
Puy ron eae 4a) A, BoC 
AB; SOD. P18 2 AB, CD 


Right. 


Equatorial, approach- 
ing bilateral, c and 
d precede. 


Right. 


Right, Cand D . 
precede. 


Left. 
Left. 
Right. 
Left. 


CD precedes. 


-I 


Or 


KOFOID: DEVELOPMENT OF LIMAX. 


period in the seventh generation. The cleavages of the following gen- 
erations are meridional or equatorial, and belong to the bilateral period. 
Another noticeable feature is the general precedence of the mesoderm- 
producing quadrant d in the cleavages of the various quartets. 

The bilateral period in the cleavage of Umbrella is not so sharply 
marked off from the spiral period as it is in Nereis ; in Umbrella spiral 
cleavage occurs as late as the ninth generation. In Nereis it ceases in 
the seventh. The quartets 5.3 and 6.2 in both forms are the ones in 
which the spiral character of the division first gives way to the merid- 
ional and equatorial cleavage, — characteristic of the bilateral period. 

The cleavage of Umbrella, like that of Nereis, presents no contradic- 
tions to the law of alternation of spirals. This striking agreement of 
Nereis, Umbrella, and Limax must far outweigh any seeming contra- 
diction arising in the work of the earlier writers upon spiral cleavage. 
It is only necessary to apply the proposed system of nomenclature to the 
careful work of Wilson and Heymons to make clear at once that the 
alternation defended holds good. The system of nomenclature employed 
in this paper facilitates the demonstration of the alternation of spirals 
in successive generations of cells ; but the alternation itself is a factor 
independent of mere names. It is the fundamental basis of the so 
called “spiral type” of cleavage. A recognition of this fact might well 
be embodied in nomenclature, and alternating cleavage substituted for 
the ambiguous and misleading term “ spiral cleavage.” 


D. The Mesoderm. 


In the forty-four-cell stage, at which the discussion of the cleavage of 
Limax was dropped, the germ layers are already differentiated. The 
quartets 7.3, 7.4, 6.3, 7.7, 7.8, 6.5, 6.6, 6.7, 6.8, are pure ectoderm, the 
quartet 7.1 and three fourths of the quartet 7.2, viz. a7?, 677, and c”?, are 
entoderm, while d‘? is the sole representative of the middle germ layer. 

The seven entoderm cells and their progeny come to lie in the region 
of the blastopore, and with the invagination are carried in to form the 
lining of the archenteron. The primary mesoblast divides bilaterally, 
i. e. in the median plane of the embryo, shortly after the forty-four-cell 
stage. The two mesoblasts retain a slight connection with the exterior, 
and at the ninety-cell stage have each divided transversely, the periph- 
eral and posterior pair of cells are the smaller, and retain a slight con- 
nection with the exterior. The next division occurs in the anterior 
pair. The cells of the mesoderm continue to multiply until there are 


“76 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


formed upon either side of the median line, extending forward from the 
posterior lip of the blastopore, two lateral bands of mesodermal elements. 
These bands are shown in longitudinal section in Figures 48, 49, and 50 
(Plate VII.) ; in transverse section, in Figures 45 and 46. In Figure 48 
the band consists of five cells, the posterior one of which in this stage 
is the largest. The band is somewhat curved, so that the anterior ends 
diverge from the more closely approximated posterior cells. In Figure 
50 the lateral band contains six cells, the two at the posterior end 
being in a mitotic state. The position of their spindles is significant of 
the manner in which the bands have arisen, viz. by proliferation 
anteriorly from the posterior cell and its products. The ‘pole cell” 
as such is not sharply distinguished from the rest of the band by its 
size, as it is in Umbrella and Nereis. The position of the nuclei of two 
of the cells is suggestive of a division in a plane coinciding with the 
axis of the band. I have however never found a spindle in a plane 
perpendicular to that axis, though spindles parallel with the axis of the 
band are frequently found. 

I have seen no evidence whatever that any of the cells of these meso- 
dermal bands are derived from any other source than the primitive 
mesoblast, d’?. It is of course impossible to prove that none of the 
cells can have come from the external layers, either by migration inward, 
or by the division of a superficial cell in a plane parallel to the surface ; 
but in the absence of any evidence that this does take place, and with 
such proof as Figure 50 (Plate VII.) gives, it seems not unreasonable 
to hold that the entire mesoderm is derived from the one cell, d’% A 
comparison of the origin and development of the mesoderm in Nereis 
and Limax shows a precisely identical origin in the two forms. In 
Nereis, however, the mesoderm shares in the generally much more 
accelerated development, so that, although it appears at about the same 
cell stage in both forms, the relative number of mesoderm cells in Nereis 
in the later stages is much greater than in a corresponding stage of 
Limax. The accelerated division of the mesodermal quadrant (D) in 
the cleavages of the different quartets, as noted by Wilson and shown 
in the table of the cleavage of Nereis, may be a manifestation of this 
same accelerating force. I have not been able to find any trace of such 
a differentiation in the cleavage of the quadrants in Limax, where there 
are no early appearing larval organs and little acceleration in the devel- 
opment of the mesoderm. On the other hand, Lillie (93) has been 
able to establish in Unio the same tendency of the quadrant D to 
precede in division; but in Unio again there is a very early develop- 


~ 2h 


= = ae 


KOFOID: DEVELOPMENT OF LIMAX. 77 


ment of larval organs. In Umbrella this tendency is not so marked, and 
here definite protoblasts are not distinguishable as early as they are 
in Nereis. 

The later history of the mesoderm will be discussed in connection 
with the subject of gastrulation and the fate of the blastopore. There 
is never developed within these mesoblast bands, at any period of their 
history, a lumen, either such as Erlanger has described for Bythinia (’92) 
and for Capulus (’92), or of any other kind. The bands later lose 
their distinctness and break up into loose mesenchyma in which it is 
no longer possible to distinguish pole cells. The mesenchyma cells 
make their way between the ectoderm and entoderm layers, and by their 
multiplication and accumulation in different regions exercise a profound 
influence upon the form of the embryo, The obliteration of the meso- 
blast bands by this process renders the determination of the relation of 
the axes of these bands to the axes of the adult very difficult. 

Inasmuch as both Erlanger (’91) and Heymons (’93) have recently 
given very full and satisfactory reviews of the conflicting literature on 
the origin of the middle germ layer in the Mollusca, it hardly seems 
necessary for me to go over the same ground. It will suffice in passing 
to call attention to the identity of my results, as to the origin of this 
layer in Limax, with those of Heymons (93) on Umbrella, Lillie (93) 
on Unio, Conklin (91, ’92) on Crepidula, Blochmann (81) on Neritina, 
and Rabl 79) on Plahorbis, making allowance of course for possible 
differences in the case of Planorbis due to reversed cleavage. It seems 
very probable that the mesoderm may have a similar origin, i. e. from 
d*?, in the Pteropods (Fol ’75 and Knipowitsch ’91), in Aplysia (Bloch- 
mann 783), and in Fulgur (MeMurrich ’86). 


E. Theoretical Considerations. 


The question as to the relation existing between the method of forma- 
tion of the mesoderm described by Erlanger for Paludina and Bythinia, 
and the type presented in Umbrella, can find its satisfactory answer only 
in an examination of these first named forms from the cytogenetic stand- 
point. As the matter stands now, we are compelled to deny the morpho- 
logical significance of the precise method of the origin of the middle 
layer, if we maintain its homology even within the group of the 
Mollusca. 

The method of origin of the mesoderm in Cyclas, as well as the 
cleavage according to Stauffacher’s description (’93), presents the mate- 


78 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


rial for an interesting comparison with that of its near ally, Unio. In the 
latter case the cleavage is spiral (Lillie ’93), and the mesoderm (adult) 
comes, as in other cases of spiral cleavage, from d™*. In Cyclas, on the 
other hand, is found a unique type of cleavage, mesenchyma appearing 
early, possibly at the seventh generation ; but the ‘‘ mesoderm,” as dis- 
tinguished from the mesenchyma cells, appears much later, and is not 
separated from the entoderm before its bilateral division. Such a case 
as this shakes one’s faith in homologies based on forms of cleavage or 
cell lineage. Indeed, it seems impossible in the face of these conflicting 
results to assign to these phases of embryonic development any definite 
phyllogenetic significance. On the other hand, the identity of the 
cleavage processes among certain of the Mollusca (Umbrella, Unio, Cre- 
pidula, Neritina, and Limax), and the similarity of cleavage in these 
to that of an entirely different group of animals, viz. the Annelids, are 
phenomena not easily banished from the thought. They must have some 
significance, some common cause. To my mind the appeal to simple me- 
chanical principles as an explanation of the phenomenon which, broadly 
speaking, we call the spiral, or alternating, type of cleavage, affords little 
satisfaction. Ifthe principle of “the resumption of the spherical form,” 
or that of “‘ minimal surfaces of contact,” prevails in one egg, why should 
it not in all eggs? We find the spiral type occurring in eggs with no, 
with little, or with much yolk, and the yolk, when present, variously 
distributed in the blastomeres; yet the spirals. occur with absolute 
certainty and in a definite manner. Other eggs, presenting apparently 
the same mechanical conditions, cleave in accordance with an entirely 
different system, radial or bilateral, in both of which adaptations to 
mutual pressure may occur without a distinct spiral. 

We can find a satisfactory explanation of the bilateral type of cleav- 
age. It is simply an accelerated victory of a force which sooner or later 
dominates every developing egg of the Bilateria. Thus it is that the 
spiral type itself gives way to the bilateral, as Wilson has so well shown 
in Nereis. 

It must be evident to all that the spiral type is very prevalent among 
the Trochozoa, i. e. among forms in which a free-swimming larva is early 
developed. 

Thus, in Nereis at the thirty-eight-cell stage, not only are the germ 
layers completely differentiated, but most of the individual blastomeres 
are set apart as protoblasts from which definite organs or parts of the 
adult body are soon to arise. This occurs about five hours after fertili- 
zation, and at ten to eleven hours after that event the larva begins to 


KOFOID: DEVELOPMENT OF LIMAX. 79 


rotate. Here we have a complete histological differentiation, while as 
yet only a comparatively small number of cells are present. Whether 
or not there exists any causal nexus between precocious development 
and the spiral type of cleavage, is a question upon which experimental 
embryology may be destined to throw some light ; as yet experimenta- 
tion has been confined to eggs having the radial or bilateral form of 
cleavage. 

The three forms of cleavage, radial, spiral, and bilateral, are undoubt- 
edly connected. Wilson (93, p. 600) has suggested that the spiral 
type is a modification of the radial, and owes its peculiarities to mechan- 
ical conditions. I would also suggest that spiral and dclateral types are 
very intimately connected. The spirally cleaving egg is essentially 
bilateral from the time that the first cleavage plane appears, and an 
inspection of the tables of the cleavage of Nereis, Umbrella, and Limax 
shows that the embryo becomes predominantly bilateral as the spiral 
cleavage fades out. In Nereis the transition from the spiral to the 
bilateral period is abrupt ; in Umbrella and Limax the two periods over- 
lap during several generations. The cleavages which succeed those of 
the spiral type are meridional and equatorial, and I believe are to be 
referred to the bilateral rather than the radial type ; indeed, in some 
cases, as in the division of 5.3 and 7.1 in Umbrella, the division ap- 
proaches very closely the typical bilateral cleavage of the tunicate egg, 
i. e. is symmetrical with reference to the median plane of the embryo. 
Wilson (’92, p. 391) has referred the meridional cleavage of 5.3 to the 
radial type. In Nereis this quartet divides before the mesoderm 
appears ; in Umbrella and Limax after it appears. When, however, in 
Nereis the quartet 7.15 divides equatorially after the mesoderm is 
formed, Wilson refers this division to the bilateral type. It seems 
to me that all these equatorial and meridional cleavages succeeding the 
spiral divisions both before and after the mesoblast appears must be 
referred to the bilateral period of the embryo and to the bilateral type 
of cleavage. 

The precise agreement of Umbrella, Nereis, and Limax in these first 
bilateral cleavages is evidently something more than mere accident. 
The meridional character of the division in two of the cases (Limax and 
Nereis) suggests the possibility of similar mechanical conditions. But 
if all the conditions in the two cases are compared more closely, it 
becomes clear that there are important differences. The cleavage in 
question takes place in Nereis at the twenty-nine-cell stage, in Limax 
at the forty-four, and a comparison of Figure 39 or Figure 41 (Plate 


80 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


VI.) with Wilson’s Plate XV. Fig. 23 shows at once the great difference 
in the shape of the egg, and the mechanical environment of the cells 
under consideration. The evidence in this case, therefore, seems to 
point to some other force than that of mechanical condition as the 
determining cause of this remarkable agreement. 

The intimate association of the spiral and bilateral types of cleavage, 
and also the prevalence of spiral cleavage in those animals possessing 
precociously developed larval forms, in which bilateral symmetry and 
histological differentiation are early impressed upon the cleaving ovum, 
suggest that the cause of spiral cleavage does not lie entirely in the 
external mechanical environment of the cells, but is, in part at least, 
to be referred to the same “morphogenic force” which produces the 
bilateral symmetry of the embryo and the adult.. That the ultimate 
fate of cells exercises a profound influence upon their cleavage is well 
shown in the precocious cleavage of the mesoderm quadrant in Nereis 
and Unio, and of the teloblasts of the larval excretory organs in 
Umbrella. It may be that in like manner spiral cleavage itself is 
but a manifestation of precocious development of the organism as a 
whole. 

It is also difficult to explain the alternation of spirals by the mechan- 
ical conditions attending their formation. A glance at the tables of 
cleavage which I have given will quickly suggest that, although we have 
the same spiral in a given division of any generation in all the eggs 
having spiral cleavage, the conditions under which the spiral is formed 
in the eggs of different animals are by no means identical. The chrono- 
logical sequence of the division of quartets in different eggs is not the 
same; neither is the distribution of the yolk, either in quantity or 
quality. The spirals however are always identical wherever they occur. 
These external mechanical conditions have doubtless a profound infln- 
ence, but are they the only or the prevailing ones? If we predicate 
this, we must maintain that the resultants of these variously combined 
mechanical influences are identical in all cases of identical spirals. Be 
the cause of the spiral what it may, the internal conditions of nuclear 
division seem to be correlated with the alternation in direction in suc- 
cessive generations. In an unimpeded field of action, the division and 
subsequent equal migration of the two daughter centrosomes would 
necessarily produce a series of cell divisions at right angles to one 
another. This element is doubtless one of the factors in that field of 
complex activities, the cleaving ovum. 


OE er aE 


OSES IF Gage Ee 


a 
i 
i 
4 
q 


a ee 


KOFOID: DEVELOPMENT OF LIMAX. 81 


F. The Cleavage Cavity. 


l. In Limaz. 


After the blastomeres have reached the widely divergent state seen 
in Plate I. Fig. 14, they begin to flatten against each other, gradually 
losing their individual spherical contour and assuming a hemispherical 
shape. This process occupied, in a case recorded, about an hour, and 
was comparatively more rapid in the latter part. It results in the ap- 
proximate restoration of the egg to the form of a single sphere. The 
superficial region of contact of the two cells appears in the living egg as 
a somewhat irregular line in the now almost obliterated furrow. Very 
soon after this process is completed there appear along this line len- 
ticular or irregular spaces, devoid of the granular structure of the proto- 
plasm, and apparently filled with a clear fluid. Deeper focusing reveals 
the fact that the centre of the apposed faces of the blastomeres is occu- 
pied by a slight cavity, wedge-shaped toward the vegetative pole, and 
broader and rounded toward the animal pole. This cavity gradually 
increases in size, the minor lenticular spaces increase also, and con- 
tiguous ones may be seen to coalesce. Finally, as the ceutral cavity 
increases more and more, and approaches the periphery of the facet of 
contact, the lenticular spaces themselves disappear, probably contributing 
their contents to the encroaching central cavity. The latter now pre- 
sents the form of a broadly lenticular clear space extending from the 


animal to the vegetative pole of the egg, and 


Figure E. 


symmetrically developed with reference to these 
poles. The two cells are thus almost com- 
pletely separated from each other by the fluid 
filling the cavity, as will be seen in the accom- 
panying Figure E, giving an optical section 
in the plane of the equator of a two-cell stage 
of Limax agrestis, showing a cleavage cavity. 


They remain in intimate connection, however, 

at the peripheral margin, but this margin of union is in some cases 
reduced to a very thin layer of protoplasm. There is apparently no 
difference in the extent of the union at the two poles. The growth 
of the cavity results in an appreciable increase in the volume of the egg, 
and its contour, as well as that of the cavity itself, is suggestive of the 
high state of tension existing in the egg as a result of this increase in 


volume. In extreme cases, as in Plate V. Fig. 34, and in Figure E, the 
VOL. XXVII. — NO. 2. 6 


82 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


cavity may attain a volume equivalent to one half or two thirds that of 
the undivided ovum. Throughout its whole history, from its inception 
to its culmination, both in the living egg and in preserved material, 
the cavity is always sharply marked off from the protoplasm of both 
cells. This is true, no matter what may be the point of view from 
which the egg is observed. Of course the boundaries of the cavity are 
indistinct where they lie oblique to the optical axis of the microscope, 
but there is always one focal plane at which the limiting “membrane” 
of the bounding cells sharply and distinctly separates the protoplasm 
from the fluid contents of the cavity, and moreover there is no trace 
whatever of any shading off of the protoplasm toward the cavity such as 
Stauffacher (93) describes in Cyclas. We are therefore compelled to 
conclude that the cavity is distinctly intercellular. 

The maximum development of the cavity is followed by a forcible 
expulsion of its contents. This takes place suddeuly, and the elimina- 
tion of the fluid may be total, or only partial. After a total elimination 
the egg resumes its original size, and tends to take again the spherical 
form. In case of a partial expulsion the cavity retains its polar diameter, 
but the antero-posterior diameter is reduced (Plate I. Figs. 5, 6). In the 
egg represented in Figure E, the spindles leading to the third generation 
were present when the cavity of the two-cell stage had reached its max- 
imum. It often happens that two or even more expulsions of the liquid 
contents of the cavity occur between the two- and the four-cell stages. 
In the series represented in Plate I. Figs. 1-7, drawn from the same egg 
kept under continuous observation, the two blastomeres had attained the 
maximum divergent or rounded condition at 11.30 a.m. At 12.35 P.M. 
they had reached the flattened or coalescent condition (Figs. 1, 2), and 
at 12.45 p.m. the central cavity had appeared (Fig. 2). This increased 
in size (Figs. 3-5), reaching a maximum at 1.30 p.M., when a partial 
expulsion occurred (Fig. 6). After this expulsion the nuclei could no 
longer be seen distinctly in the living egg. The cavity again increased 
in volume (Figs. 6, 7), and at 2.02 p.m. a second and total elimination 
took place coincidently with the division into four cells; this was accom- 
plished, i. e. the furrows had reached the vegetative pole, at 2.05 P.M. 

It is not always possible to determine the point at which the fluid 
contents are ejected. When a sudden reduction in the size of the cavity 
is noticed, there is sometimes visible in the albumen adjacent to the 
cleavage furrow a small sphere of transparent matter differing in its 
refractive index from the surrounding albumen into which it very quickly 
merges. When, however, the reduction in the size of the cavity is 


a a eee eee 


KOFOID: DEVELOPMENT OF LIMAX. 83 


gradually accomplished, occupying in one instance recorded about five 
minutes, no trace of the extruded liquid is visible. I have observed in 
the two-cell stage the expulsion of the liquid at both animal and vegeta- 
tive poles, but never at both poles of an egg at the same time. Warneck 
(750) and Fol (80) both state that the contents of the cavity are ex- 
pelled at the vegetative pole. This is certainly by no means constant, 
and I am inclined to believe that in a majority of cases, especially in the 
later stages, the elimination takes place at the animal pole of the egg. 

This ephemeral cleavage cavity is not confined in Limax to the two- 
cell stage, but is equally prominent in the stages immediately following. 
The passage of the egg from the two- to the four-cell stage may be 
accompanied by an incomplete elimination of the contents, for I have 
often observed cases where a small cavity persists throughout the 
progress of this cleavage. 

Figures 8-13 (Plate I.) show the history of the cleavage cavity in a 
different egg from the one observed during the two- to four-cell stage. 
At 3.15 p. mM. there was no trace of any cleavage cavity, and the second 
cleavage furrow had almost reached the vegetative pole. Half an hour 
later the characteristic four-cell condition had been reached (Fig. 8), 
and in ten minutes more a cleavage cavity of considerable volume was 
developed in the vertical axis of the egg. This continued to increase in 
size until 4.45 p. m. (Figs. 9-11), when a total expulsion of the contents 
occurred, occupying not more than thirty seconds (Fig. 12). The nuclei 
at this period were at the amphiaster stage. Within fifteen minutes a 
new cavity had appeared in the now elongated vertical axis of the egg. 
This cavity was at first very narrow and extended almost from pole to 
pole. It increased slowly in volume, but was not wholly obliterated at 
the division into eight cells, which occurred at 5.38-5.45 p. mM. (Fig. 13). 
It is not at all unusual to see the total elimination of the contents of 
the cavity at the division into eight cells, but the occurrence is not 
constant. The configuration of the cavity of the four-cell stage as 
viewed from the animal pole is shown in Plate II. Fig. 17. It is almost 
rhomboidal in outline ; the angles lie at the cleavage planes, and the 
sides are curved with the convexity next the cavity. It is probable that 
a partial expulsion, or perhaps a total one, has already occurred, for 
the cavity was not very large and the nuclei were in the early phase 
of metakinesis when the egg was killed. When the cavity is at its 
maximum it assumes very nearly a spherical shape, i. e. the bounding 
cells are concave toward the cavity, and they present more nearly the 
character of a wall of uniform thickness (Fig. E, p. 81). No case has 


84 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


come under my observation where a nucleus projects into the cavity, as 
Stauffacher ('93) figures it in his Tafel XIIT. Fig. 19 a. ‘ 

An interesting condition is found in Plate IT. Fig. 16, in which the second 
cleavage furrow is almost completed. The cavity appears to have been 
divided into two parts by the recent cleavage furrow, and now consists of 
two large lenticular spaces, one between the cells A and JD, the other 
between 4 and C, i. e. both spaces are in the first cleavage furrow. The 
first appears to lie mainly in the cell A, but this is due to the fact that 
A lies slightly above and upon D. The cavity between & and C has 
several secondary contributory spaces lying superticially to it iu the 
furrow at the animal pole. 

The cavity of the eight-cell and later stages differs from that of the 
two-cell stage in that it is situated nearer the animal than the vegetative 
pole of the egg. This is correlated with the size of the two quartets of 
the fourth generation, Plate Ii. Figs. 20 and 21, and may be the occa- 
sion of the frequent escape of the fluid contents at the animal pole. 

It is not necessary to follow in detail the phenomena which attend 
the further history of the cleavage cavity, as it would be in the main a 
repetition of the description of that of the earlier stages. I shall merely 
call attention to certain features of the cavity which are of especial 
interest. 

An examination of a large number of eggs in the living state, as well 
as killed and hardened material studied both ¢x toto and in sections, has 
led me to the conclusion that this ephemeral and recurrent phase of the 
cleavage cavity or blastoccel continues until a late stage, even to the 
period of gastrulation. That its appearance is not due to a pathological 
condition of the embryo is shown by the prevalence of the same phenom- 
enon in eggs collected in the natural environment of the slug, as well as 
by the development of normal embryos from vacuolated eggs. It may 
be that confinement conduces to the presence of the ephemeral cavity 
in its various forms, but I have no direct evidence that such is the 
ease. 


> 


Eggs presenting the maximum development of the cleavage cavity in 
the later stages are with great difficulty freed intact from their enve- 
lopes and require especial care in the subsequent treatment with reagents. 
On Plate V. (Figs. 33 and 34) is figured an egg of twenty-four cells 
with a well developed cleavage cavity. The nuclei are all in a quiescent 
state, and the cells form a wall of such uniform thickness that it was 
only after repeated trials that the vegetative pole of the egg could be 
determined. The cavity is so large that the facets of contact are very 


KOFOID: DEVELOPMENT OF LIMAX. 85 


narrow, and the individual cells are in optical section somewhat lozenge- 
shaped. The cell a’ (Fig. 34) presents a curious bud-like process 
extending into the central cavity, and the superficial extent of the cell 
is somewhat less than that of the other members of the same quartet. 
This process suggests the mesenchyma cells which Stauffacher (’93) 
figures in his Tafel XIV. Figg. 25a and 25c, but in this projection 
there is not the least trace of any nuclear structure, and it is probably 
a mere amceboid outgrowth of no permanent significance. 

A comparison of the computed volumes of the whole egg, of its 
cavity, and of the protoplasmic portion, with the volume of another 
egg (Plate IV. Fig. 27) of the same stage but having no cavity, brings 
out the following results. The whole egg has 429 units of volume, of 
which 188 represent that of the cavity, 241 that of the blastula wall, 
while the twenty-four-cell stage of average size (ig. 27) has a volume 
of only 126 units, These figures assume the perfect sphericity of the 
objects measured, and are therefore only approximately correct ; still 
they show that the first egg, though a large one, is within the limits 
of variation in size, and that the cavity is larger than the average egg, 
but not so voluminous as the substance of the egg which contains it. 
It is also suggested, in view of the large size of the egg, that the cavity 
has not been developed to any great extent at the expense of the volume 
of the protoplasm of the egg. ‘There can be no question that this egg 
presents the condition of a typical * blastula” with a typical “ cleavage 
cavity’ or blastoceel. Indeed, Rabl could not have found for Haeckel 
and his Gastraa Theory a better illustration among mollusks of the 
“morula” and “blastula” stages than these two twenty-four-cell stages 
(Plate IV. Fig. 27 and Plate V. Fig. 34); for the first contains no 
cavity whatever, and the latter has its cells arranged in a single layer 
about a cavity. On the other hand, if we accept the limitation set upon 
our usage of the term cleavage cavity by Stauffacher in his recent paper 
(93), we shall be compelled, in view of the fact that the cavity is sooner 
or later entirely eliminated, to call this beautiful example of a cleavage 
cavity simply “ ein heller Raum.” 

It is difficult to establish any regularity or uniformity in the sequence 
of the phases of the cavity in these later stages of cleavage. When we 
examine other eggs in the twenty-four-cell stage we meet with different 
and by no means constant conditions. The twenty-four-cell stage repre- 
sented in Plate IV. Fig. 31, shows no trace whatever of a cavity ; while 
Figure 28, also a twenty-four-cell stage, shows at the animal pole a 
number of lacune or intercellular vacuoles between the cells of the 


86 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


apical quartet, a°*—d°* and their neighbors. An especially large 
vacuole is formed immediately at the animal pole. 

Inasmuch as the vacuolation of the animal half of the egg is an impor- 
tant and very prevalent occurrence in the later stages in the cleavage of 
Limax it deserves a detailed description. In surface view these cavi- 
ties are seen to be arranged in general along the line of the cell bounda- 
ries, which they obscure to such an extent that the superficial margins 
of the facets of contact are detected only by careful focusing upon the 
immediate surface of the egg properly illuminated. As scon as the plane 
of the focus is lowered toward the level of the nuclei, the boundaries are 
at once lost and nothing but a clear space can be found. The proto- 
plasm peripheral to the cavity is therefore comparatively thin, and does 
not present the granular structure of the deeper lying regions. The 
cavities in many cases extend laterally upon either side some distance 
from the superficial line of contact of the two cells, and sometimes, as in 
the cell c®*, Figure 28, they even lie between the nucleus and the 
external surface of the cell. In all cases it is possible to detect a sharp 
and definite boundary to these cavities, when the egg is so oriented as 
to bring the margin of the cavity into the proper relation to the optical 
axis of the microscope. These boundaries have the same appearance in 
whole preparations and in sections that cell boundaries have, and indeed 
I believe that they are cell ‘‘membranes,” and that the cavities are 
strictly intercellular. That part of the facet of contact lying peripherad 
to the cavity is not continuous through the cavity with the part centrad 
(Plate III. Figs. 24, 25), but is in direct continuity with the wall of the 
cavity. This seems to me to be indisputable proof that these vacuoles 
are intercellular structures, just as the lenticular spaces and central 
cavity of the earlier stages of cleavage and the large cavity of the twenty- 
four-cell stage are. The question as to whether these should be called 
the cleavage cavity will be discussed later. 

The appearance of these cavities in section is shown in Figures 24 and 
25 (Plate III.). The egg here represented is a very small one, only 
80 w in diameter, and is shown 7 toto in Figure 23. It has just been 
derived from the sixteen-cell stage by the division of the quartets 5.1 and 
5.2. Traces of this division can still be seen in the derived quartets 
6.1, 6.2, and 6.3, 6.4. The sections were cut obliquely to the vertical 
axis, and so directed as to cut longitudinally the remnants of the spindles 
in one of the quadrants of the quartets 6.3 and 6.4. There is a medium- 
sized central cavity, which, owing to the recent division and consequent 
rounded condition of the cells concerned, lies nearer the vegetative pole. 


KOFOID: DEVELOPMENT OF LIMAX. 87 


The section shown in Figure 25 cuts the cells of the animal pole ob- 
liquely, and thus exaggerates their relative size somewhat. In addition 
to the central cavity, there are a number of smaller cavities between the 
cells of the animal pole. Their relation to the cell boundaries can in 
every case be readily determined in the sections. The larger cavity 
x of Figure 24 is between two cells whose facet of contact lies parallel 
to the plane of the section ; the cavity therefore appears to traverse a 
cell, thongh in reality it does not. In the figure it is in direct continuity 
with cavities which are readily seen to be intercellular. These latter 
cavities appear lenticular in cross section, but they are themselves elon- 
gated as the cavity x is. There are, in addition to the intercellular 
cavities just described, two others (vac., Figs. 24 and 25), which seem to 
be intracellular). They are both near the central cavity, though not 
in direct contact with the cell membrane in any direction. They are 
both approximately spherical in form, and neither has the sharp and 
definite outline separating it from the protoplasm of the cell that the 
intercellular spaces just described have. Their form, position, and limits 
thus indicate their intracellular nature. They probably are merely intra- 
cellular vacuoles. Their position is suggestive of their fate. They lie 
very near the central cavity, and it may be that their contents ulti- 
mately find their way into it by osmosis, or less probably by rupture of 
the “cell wall.” 

The fate of the fluid eliminated from the lenticular spaces of the earlier 
stages, and from the intercellular spaces of the later stages, is a difficult 
matter to determine. Direct observation gives negative results, for 
although these cavities in the early stages can be seen to develop and 
disappear, it is impossible to say what becomes of their contents. I have 
never seen avy evidence of the extrusion of their contents from the egg, 
but this might escape observation, for the cavities are small and their 
contents are transparent, and it is often difficult to determine the point 
at which the contents even of the large cavity are expelled. 

There are, however, some facts which lend support to the view that 
these lenticular spaces of the early stages, and perhaps also the inter- 
cellular spaces of the later stages, contribute directly to the increase of 
the volume of the central cavity. The nuclear conditions of Figures 
23-25 (Plate III.) show beyond question that this twenty-four-cell stage 
is younger than that of Plate V. Fig. 34, where the larger cleavage 
cavity is shown. It seems reasonable to derive the conditions of the 
older stage from those of the younger. The main difference between 
the two stages lies in the increased size of the cleavage cavity and the 


88 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


absence of the secondary intercellular cavities in the older egg. It 
seems probable that, as the central cavity grows in volume and the 
facets of contact diminish in size, the central cavity extends to these 
secondary cavities and fuses with them, and that thus all portions of the 
surfaces of the cell, except its exterior one, may contribute secretions to 
the central cavity. The immediate proximity of several of these sec- 
ondary cavities to the central cavity in Figures 24 and 25 (Plate III.) 
suggests that the former may even actually move toward the central 
cavity. The direction of the motion is merely a question of the direc- 
tion of least resistance ; it is difficult to explain the development of such 
a large cavity as that of Figure 34 (Plate V.) and the subsequent forci- 
ble expulsion of its contents, and the immediate restoration of the egg 
to a solid spherical mass, without admitting the existence of a consider- 
able force, tending to preserve intact the contour of the egg, and resist- 
ing the increasing tension brought about by the enlargement of the 
central cavity. If the contents of these smaller cavities are eliminated 
to the exterior, why should not those of the large cavity, whose tension 
must be proportionally ‘greater, be eliminated at the same time? There 
is a point, however, beyond which the increase in the size of the cen- 
tral cavity cannot go. The outer wall yields to the pressure, and the 
imprisoned fluid escapes. I have found no trace of an egg membrane, 
such as Gegenbaur (’52) has described for the egg of Limax agrestis: 
“ Es besitzt eine Zellmembran, die besonders durch langere Einwirkung 
von Wasser deutlich erkennbar wird.” There is no evidence that there 
is anything more than the ordinary clear stratum of protoplasm at 
the exterior of the egg. In this my observations are in accord with 


those of Dr. Mark upon Limax campestris (81). None of these secon- ~ 


dary cavities or lenticular spaces were observed in the eggs which have 
the maximum central cavity, neither have they been seen at the time of 
the expulsion of the contents of the cavity, even when that takes place 
eradually. They are associated with the growth rather than with the 
disappearance of the central cavity. In view of these facts, it seems to 
me that we are justified in concluding that, in some cases at least, the 
lenticular and the secondary intercellular spaces contribute to the 
increase of a central cavity. 

As has been stated already, these secondary intercellular spaces often 
form at the animal pole of the egg, while not a trace of them can be 
found at the vegetative pole. They may present the appearance of an 
anastomosing network of irregular vessel-like structures between the 
cells of that pole of the egg, as in Plate VI. Fig. 39. It hardly seems 


oD? 


4 KOFOID: DEVELOPMENT OF LIMAX. 89 


; possible that a histological differentiation can have already taken place 
between the two poles of the egg whereby the cells of the animal pole 
are set apart to perform an excretory function. This is rendered still 
more doubtful by the frequent occurrence of eggs in which these secon- 
dary intercellular spaces have reached an enormous development at both 
poles, in fact throughout the whole egg. This condition may occur as 
early as the twenty-four-cell stage. In such eggs there is never any 
distinct central cavity present; it becomes difficult in such cases to 
locate cell boundaries and the reijation of nuclei to them. In Plate III. 
Fig. 26, is shown a transverse section of such an egg containing more 
than one hundred cells. In stainability and nuclear conditions this is 
not essentially different from other eggs ; several cells of this egg are in 
a mitotic state ; I therefore believe such eggs to be normal. As can be 
seen in the figure, the three germ layers are present, and the vacuolation 
surrounds the cells of all three layers indifferently. There is no central 
cavity, and the three layers retain their connection with one another. 
Indeed, this condition is very suggestive of that found in the gastrula at 
the time when the head-vesicle is beginning to develop and the entoder- 
~ mal cells are sending out long processes into the fluid-filled space toward 
. the cells of the other layers. It seems therefore no misuse of terms to 
_ designate the intercellular spaces in both cases as the primary body 
_ eavity, which throughout the period of segmentation is also the cleavage 
cavity. The condition represented in the figure is ephemeral and the 
extrusion of the liqnid contents may take place without the formation 
of a spherical central cavity. The spaces seem to be thoroughly 
connected with one another and when some point on the periphery 
of the egg yields to the pressure, the fluid is probably in large part 
eliminated. 
The occurrence of a single distinct central cavity is shown in Figure 
_ 47, section of an embryo of eighty cells, and likewise in Figures 48 
_ and 49 (Plate VII.), where the embryo has assumed the flattened shape 
_ characteristic of the stage preceding gastrulation. In this egg the cavity 
is small and lies between the ectoderm, the entoderm, and the bilaterally 
placed mesoderm bands. There is no trace of any cavity in the meso- 
-derm. In Figure 54 the cavity occupies a position at the posterior end 
of the blastopore, and, as in the preceding stage, lies next to the ecto- 
derm on the dorsal side of the embryo. I have found many embryos, 
not figured, which have this definitely limited central cavity. In no 
_ case, however, has it attained the size of the cavity in the twenty- 
four-cell stage shown in Plate V. Fig. 34. On the other hand, a large 


5 ta 


90 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


part of the embryos examined have either the intercellular vacuolation 
or no trace of any cavity whatever. 

What is the morphological and what the physiological significance of 
the phenomena with which we have been dealing? Are these cavities 
all simply different phases of one and the same thing, — an ephemeral 
recurrent cleavage cavity, — or do they differ among themselves, and are 
they to be considered as different from the cleavage cavity? Do they 
bear any relation to the conditions under which the egg develops? My 
work has left no doubt in my own mind that they all belong to the same 
and that they are 
perhaps intimately connected with the conditions in which the embryo 
develops. I shall refer to these points again after reviewing the litera- 
ture of the subject. 

The question might well be raised as to whether the term cleavage 
cavity should be used to designate the phenomena which we have dis- 
cussed. If we are to apply this term to that continuous, persistent 
cavity into which the invagination resulting in gastrulation takes place, 


category, — modifications of the cleavage cavity, 


and that only, then we assign to the term an unduly limited morpho- 
logical significance, suggested by the Gastraea Theory of Haeckel. Then 
this ephemeral cavity in Limax is not a cleavage cavity, and we must 
coin some new term to distinguish it, such, for example, as excretory 
cavity. If, on the other hand, we recognize the physiological importance 
of this and other cleavage cavities, while not denying their morphological 
significance, and bear in mind also the constant intercellular nature of the 
phenomena in question, it is in my opinion perfectly legitimate to desig- 
nate by the term cleavage cavity any and all of the protean forms which 
the intercellular space assumes in Limax. The matter of terms is, how- 
ever, a minor point, the unity of the phenomena is the important one. 
There remains one more question of interest. Is there any relation 
between the stages of cleavage and the recurrence of the cavity? 
Warneck, in 1850, stated that the cavity reaches its greatest develop- 


ment contemporarily with the “Maximum der Entwickelung der Kerne.” — 


My own observations do not show that this is always the case. In 
Figure E, the two cells enclosing the large cavity contain, not nuclei with 
distinct membranes, but spindles. There is a mechanical cause for the 
elimination of the contents of the cavity at the period when the cells 
assume a rounded condition at the close of cleavage. The facets of 
contact are then much reduced, and the resistance at the periphery of 
the egg to the expulsion of the fluid is more readily overcome. It may 
also be that the periods of great activity in the cells at the time of 


—— 


——— 


— ae ee 


KOFOID: DEVELOPMENT OF LIMAX. 91 


division are periods at which the osmotic processes reach a maximum, 
and thus the cleavage cavity may grow rapidly at this time. My obser- 
vations on living eggs show that the period immediately preceding di- 
vision is that of the most rapid growth of the cavity. It is not an 
uncommon thing to find in the later stages neighboring cells in a 
mitotic state enclosing a lenticular space between them. These two 
causes may result in producing in some cases, during the early stages of 
cleavage, an apparent rhythm between the nuclear conditions and the 
periods of expulsion. There is, however, much variation in these early 
stages, and it is impossible to establish in them any such constant 
correlation as Warneck has indicated. 


2. Lnterature. 
AMPHINEURA. 


No mention is made of any cleavage cavity in the development of 
Dondersia, as described by Pruvot (90). Kowalevsky (’83) does not 
discuss the subject in Chiton, but Metcalf (93) describes the cleavage 
cavity as already formed at the four-cell stage. No statement is made 
_ about its subsequent disappearance. 


LAMELLIBRANCHIATA. 


4 I. Marine Forms. 


Lovén (’48) does not figure a segmentation cavity in either Modiolaria 
_ orCardium. Barrois (79) makes no reference to any segmentation cavity 
q in Mytilus, though his Plate XII. Fig. 16, if it represents a section, shows 
_ such a cavity. He distinctly states that the segmentation produces a 
_ body considerably larger than the original ovum. He also notes in the 
’ two-cell stage the appearance, in one instance, of lenticular refractive 
_ bodies apparently identical with those figured by Bobretsky as found in 
Nassa mutabilis. These bodies are adjacent to the furrow separating 
the micromere and macromere of the two-cell stage, and may be due to 
_ a highly refractive secretion accumulated in these regions. 
Brooks (’80*) describes in Ostrea Virginiana a transparent cavity sepa- 
rating the ectoderm from the macromere in dead eggs at a stage when 


the macromere is almost covered by the very large number of ectoderm 
cells present. He does not regard this space as normal, since the macro- 
“mere seems in living eggs to be in contact with the outer layer, and 
_ there is no indication of a segmentation cavity. It is only concerning a 
later stage, when the macromere has divided into a number of entoderm 


92 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


cells, and the embryo is much flattened in the dorso-ventral direction, that 
he says a distinct ‘segmentation cavity, or more properly a body cavity, 
is now clearly visible” between the ectoderm and the entoderm. Horst 
(82) says, “It is not possible to demonstrate the existence of a true 
cleavage cavity in the oyster.” 

Hatschek (’80) finds that one of the peculiarities of the development 
of Teredo is ‘der giinzliche Mangel der Furchungshohle.” 


Il. F'resh- Water Forms. 


Forel (’68, p. 14) called attention to the fact that the “ yolk” in Unio 
later becomes clear and transparent, but he failed to interpret this 
appearance as a cavity. It remained for Flemming (’75) to show in 
Anodonta the presence of a cleavage cavity as early as the two-cell stage, 
to establish its recurrent character, and to assert its equivalency to the 
cleavage cavity of the later stages. He notes its formation as a lens- 
shaped cavity between the macromere and micromere of the two-cell 
stage ; its disappearance with approaching cleavage; its reappearance 
in the four-cell stage, and its subsequent disappearance before the next 
cleavage begins. He also notes its relatively large volume in a stage 
when there are from six to ten micromeres, but he does not describe 
any further obliterations of the cavity. The fact that the earlier cavities 
are obliterated does not raise the question with him as to whether they 
should be regarded as cleavage cavities or not. 

Rabl (’76) has observed a similar phenomenon in Unio pictorum. He 
describes the cavity as a transparent protoplasmic layer with few yolk 
granules between the cells of the two-cell stage. He contends, however, 
that it is not the beginning of the cleavage cavity, as Flemming had 
maintained, and calls attention to the fact that similar regions, free 
from granular structures, between two or more cleavage spheres, are 
met with in the embryos of other animals, as, for example, in many Gas- 
teropods, where the cleavage cavity appears later than it does in Unio. 
At the four-cell stage, however, he recognizes “die erste Anlage der 
Furchungshohle,” but does not speak of any reduction or disappearance 
of this cavity in later stages, though some of his figures suggest it. 

Korschelt (91) speaks of an expansion of the primitively narrow seg- 
mentation cavity of Dreissena, during which the embryo, which has 
reached the gastrula stage, assumes a roundish oval shape. No mention 
is made of the ephemeral or recurrent character of this primitive cavity. 
It is a matter of considerable interest to see that Dreissena, which is a 
“near ally of the common mussel,” and is probably a recent migrant 


KOFOID: DEVELOPMENT OF LIMAX. 93 


into a fresh-water environment, still retains the free-swimming larval 
stage characteristic of marine forms. It has acquired, however, the 
primitive segmentation cavity” found in the fresh-water Lamelli- 
 branchs, but not definitely known to be present in the marine forms. 
Lankester (’74) does not refer to the cleavage cavity of Pisidium, nor 
does he figure it except in comparatively late stages of development. 
Von Jhering (’76) speaks of the three or four small cells in Cyclas, 
whose progeny grow around the solid mass of the two large cells, and 


of the later appearance of a cavity in the centre of this mass. Ziegler 
(’85) finds a cavity in the thirteen-cell stage of Cyclas, but indicates no 
cavity in the two earlier stages that he figures. 

The latest, and by far the most important, contribution to our knowl- 
edge of the cleavage cavity is that of Stauffacher (93) upon Cyclas 
cornea. The formation of a “true” cleavage cavity takes place at the 
thirteen-cell stage by the gradual elevation of the cap of ectoderm cells 
from the macromere to which they had been closely applied, resulting 
in the development of a sharply defined space between the macromere 
and its derivatives. ‘This cavity persists and increases in size until it 
ultimately becomes the relatively very large cavity of the blastula stage. 
_ In addition to this cavity, which he regards as persistent from the 
 thirteen-cell stage on, Stauffacher finds in the two-cell stage a structure 
which he regards as similar to that observed by Flemming in Anodonta 
and by Rabl in Unio. He describes it as a disproportionately large 
_ space, entirely unstainable, zz the smaller of the two spheres, exactly in 
_ the region where they are in contact. The cavity is filled with a fluid 
free from granules. The protoplasmic part of the cell, which forms the 
peripheral layer and contains the nucleus, merges very gradually into 
_ this fluid-filled space. On the side of the macromere this space is 
sharply and definitely limited. It seems from his description that this 
space is regarded by him as lying zm the smaller cell, i. e. intracellular, 
though he does not distinctly designate it as such. The interpretation of 
this space and its later history are best given in his own words (Joc. cit., 
p- 211): “Es fallt bei Cyclas nicht schwer, den unumstosslichen Beweis 
zu erbringen dass der genannte helle Raum in der That nichts mit einer 
-Furchungshohle zu thun hat. Dieselbe kérnerlose Partie ndmlich, die 
wir auf dem zweizelligen Stadium antreffen, ist zwar auch auf dem drei- 
zelligen Stadium noch vorhanden, aber schon bei der Bildung der vierten 
Furchungskugel wird sie bedeutend reduziert und verschwindet schliess- 
lich ganz. Dagegen entstehen im weiteren Verlauf der Entwickelung 
zwischen der grossen Mutterzelle und ihren jeweiligen letzten Abstam- 


94 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


mungsprodukten neue solche Partien. Die eigentliche Furchungshéhle 
tritt bei Cyclas, wie wir sehen werden, erst in bedeutend hoheren 
Furchungsstadien auf.” 

A cavity similar to that of the two-cell stage is figured for the four-, 
five-, six-, seven-, nine-, and twelve-cell stages, occurring always between 
the macromere and its most recent products. This cavity becomes suc- 
cessively smaller from the four- or five-cell stage until we reach the 
relatively small cavity of the twelve-cell stage. It is always sharply 
limited from the macromere, and often presents on the side next the 
most recent micromere, or its products, the gradual merging into the 
granular protoplasm noted in the two-cell stage. That is to say, here, 
as there, the inference is that the cavity may be regarded as an intra- 
cellular space. The fluid which fills this decreasing cavity he thinks is 
absorbed in large part by the macromere, and perhaps to a less extent 
by the micromeres, and that it does not pass out of the cavity through 
the egy membrane. After this fluid-filled space has disappeared from 
between the earlier formed micromeres, m1, m?, m®, m*, and the macro- 
mere, the micromeres in question apply themselves closely to the 
macromere in a way that suggests the fusion of micromeres with the 
macromere noted, as by Lovén (49) in marine Lamellibranchs, and by 
Bobretsky (’77) in Nassa. With regard to the interesting phenomenon 
of fusion described by these authors, Stauffacher makes the following 
suggestion: “ Es erscheint mir nicht unwahrscheinlich dass vielleicht in 
allen den Fiillen, wo ein nachtriigliches Abflachen der kleineren Zellen 
konstatiert wurde, auf giinstigen Prepiiraten auch der helle Raum 
zwischen den Furchungskugeln hiitte nachgewiesen worden konnen, der 
durch sein Verschwindung das Anschmiegen der Mikromeren méglicher- 
weise bedingt.” ‘ Der helle Raum ” has, however, never been recorded 
by any investigator of these forms; furthermore, the fusion in some 
eases (and these are the most marked cases of fusion) consists in the 
reunion of the more richly protoplasmic part of the macromere with 
the more passive yolk-bearing portion, from which it had abstricted 
itself at the time of nuclear division. 

Neither Bobretsky (77) nor Brooks (’80) figures a nucleus in the 
“ macromere” with which the micromere so completely fuses ; and it 
seems hardly possible that in these cases the disappearance of a cavity 
can have anything at all to do with the phenomenon of fusion. There 
are moreover some objections to the view that in the two-cell stage the 
cavity lies within the cell, and to the inference that it is essentially of 
that nature in the later stages. Stauffacher himself does not emphasize, 


he 


KOFOID: DEVELOPMENT OF LIMAX. 95 


or even clearly present this view, though he repeatedly calls attention to 
the lack of a sharp differentiation of the cavity from the protoplasm of 
the most recent derivative or derivatives of the macromere. ‘The fact that 
this gradual transition is shown toward two cells, as in his Taf. XII. 
Figg. 14 a-g, and-Taf. XIII. Fig. 18 6, militates against the view that the 
cavity is intracellular. It may well be that the yolk-laden macromere, 
on account of its different stainability, is more sharply marked off from 
the cavity than the protoplasmic micromere ; but is it not possible that 
the gradual transition of the granular protoplasm of the adjoining cell 
into the clear space of the cavity is in most, if not all, of the cases figured 
by Stauffacher due to oblique sections of the limiting membrane? His 
figures of the whole egg are made from reconstructions on glass plates, 
and in them the outlines of the cavities are not distinctly traced. In 
most cases he has not indicated the planes of the sections which he 
figures ; these must therefore be inferred from the position of the nuclei. 
Such inferences, however, lead one irresistibly to the conclusion that the 
sections must meet the boundary of the cavity obliquely wherever its 
outline appears indistinct ; e. g. Taf. XII. Figg. 14 a-g; Taf. XIII. Figg. 
18a and b. On the other hand, sections which appear to strike the 
cavity perpendicularly, as in Taf. XII. Figg. 15 a and 6, 16a and 8, and 
17a, all show a much more distinctly marked separation of the proto- 
plasm of the cells from the cavity, and in some cases this demarcation is 
as definite on the side of the most recent micromere as it is upon that 
of the macromere. In case this explanation should prove valid, we shall 
have in Cyclas, as in Limax, an intercellular cavity appearing at the 
two-cell stage, and recurring in the later stages of cleavage. 

I cannot agree with Stauffacher’s view that this primitive “heller 
Raum” has nothing whatever to do with the true cleavage cavity. It 
is not established even by the facts found by him in Cyclas ; much less 
by a comparison with other forms presenting a similar phenomenon. 
His observations are confined to killed, preserved, and hardened material 
of very limited amount. He had in some cases not more than one series 
of sections of each cleavage stage ; of the three-cell stage seven series, of 
the four-cell stage six series. He has not been able to examine the eggs 
in the living state, or in whole preparations. Thus he has been deprived 
of most valuable assistance in determining the origin, definite bounda- 
ries, successive phases, ultimate fate, and relationships of this “ heller 
Raum,” whose claim to the title of cleavage cavity he so summarily 
dismisses. The “ unumstosslich Beweis” which he brings forward to 
support the view he advances is, that the “heller Raum” finally dis- 


96 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


appears entirely. But his figures and descriptions show that this clear 
area merely occupies a different position with reference to the first 
micromeres, not that it entirely disappears. Every stage that he figures, 
from the two- to the thirteen-cell stage, where, upon his interpretation, 
the true cleavage cavity first appears, contains a cavity. He brings 
forward no proof to show that these may not be continuous both with 
one another and with the cavity of the thirteen-cell stage, which is in his 
view the true cleavage cavity. It seems to me, then, that his own evi- 
dence does not conclusively sustain the view that this primitive cavity 
is not a true cleavage cavity, as he himself has defined it. Indeed, we 
should expect that in Cyclas, as in other fresh-water mollusks, there 
might be an entire elimination of the cavity at intervals, though he has 
not proved it. Even if we grant that in Cyclas the primitive cavity is 
eliminated, we have still the important point to consider whether or not 
such an elimination constitutes a valid ground for removing the “ heller 
Raum” from the category of cleavage cavities. A comparison of the 
phenomena in Cyclas with those presented in such a form as Limax 
would seem to indicate that we are dealing here, as there, with an 
ephemeral recurrent cleavage cavity present at the very beginning of 
segmentation. 


ScAPHOPODA. 


Kowalevsky (’83) finds a definite cavity appearing in Dentalium as 
early as the eight-cell stage. This gradually increases in size, forming 
quite a large cavity at the time of gastrulation. 


PROSOBRANCHIATA. 


I. Marine Forms. 

An examiuation of the literature of Prosobranch development shows 
an almost entire absence of references to a cleavage cavity. The few 
allusions that exist are concerned with the cavity that appears com- 
paratively late in the period of cleavage. 

Bobretsky (’77) finds a cleavage cavity in Nassa mutabilis at the 
thirty-six-cell stage. Although the alternation of the rounded with 
the flattened conditions of the cells in cleavage is quite prominent in 
Nassa, no cavity is noted as occurring between the fused cleavage 
spheres. 

MeMurrich (’86, p. 412) makes the following statement with regard to 
the segmentation cavity in Fulgur: “To one side of the blastoderm and 
below it a more or less distinct cavity is to be seen, containing granular 


, ie 


KOFOID: DEVELOPMENT OF LIMAX. 97 


matter. It is possible that this may represent the segmentation cavity, 
though it does not appear to be present in all cases.” This is ina stage 
preceding the formation of the “sixth generation ” of micromeres and 
the appearance of the mesoderm. Brooks (’78) figures at a late stage a 
cavity in Urosalpinx similar to that found by Bobretsky in a late stage 
of Nassa. Patten (’86) does not figure any segmentation cavity in 
Patella, but in the later stages, before gastrulation, a medium-sized 
central cavity is present. Conklin (’91) finds in Crepidula, “at an early 
period, a trace of a segmentation cavity, which, however, is soon 
obliterated.” 
Il. Fresh-Water Forms. 

Blochmann (’81) does not discuss the subject of the cleavage cavity 
in Neritina, but it is evident from his figures that, if it is present at all 
in the earlier stages, it does not attain a great size. Neritina contains 
a large amount of yolk, and this may have some effect on the cleavage 
cavity. In the late stages a small cavity appears between the ectoderm 
and the macromeres. 

No mention is made by Sarasin (’82) of a cleavage cavity in Bithynia 
until the close of the cleavage period. Sections of the early stages were 
not cut. Erlanger (92) finds a large cavity present at the close of seg- 
mentation, i. e. before the formation of the mesoderm and when the 
blastomere contains, according to his estimate, at least sixty cells. In 
Paludina vivipara, Lankester (’76) finds in a late stage of cleavage “a 
central space or cleavage cavity.” A cavity of considerable size is also 
figured by him as present at the time of gastrulation. On the other 
hand, neither Biitschli ?77) nor Blochmann (’83) succeeded in finding 
in this species any cavity in the cleavage stages examined by them, nor 
more than a mere slit-like cavity between the layers at the time of 
gastrulation. Erlanger (’91), however, finds a large cavity in the 
gastrula stage, and it is into this cavity that the mesodermal pockets 
described by him are evaginated. If Paludina has an ephemeral 
recurrent cavity similar to that of Limax, the apparently discordant 
observations of Lankester, Biitschli, and Blochmann would be easily 
harmonized. 

I have myself watched the cleavage of the eggs of Amnicola limosa, 
and find that they present a typical recurrent cavity, precisely like that 
of the fresh-water pulmonates. The eggs of Amnicola are enclosed in 
capsules and are surrounded by a jelly-like albumen. They contain a 
small amount of yolk, and cleavage is not so unequal as it is in 
Neritina. 

VOL. XXVII.— NO. 2. 7 


98 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


OPISTHOBRANCHIATA. 


There seems to be an entire absence of references to the presence of a 
cleavage cavity in the development of this group. 

Heymons (’93) found in Umbrella no trace whatever of a cavity at 
any period up to the formation of the larval stage. 


PTEROPODA. 


Fol (75) says of the two-cell stage of the Pteropods, ‘“ Mais il ne se 
produit pas ici, comme chez le Lymnée et la Limace, ou comme chez les 
Geryonides, des vacuoles entre les cellules.” Nor is a cleavage cavity 
described by him for the later stages. Knipowitsch (’91) mentions a 
“spaltformige und nicht immer deutlich wahrnehmbare Furchungs- 
hohle,” into which the mesodermal cells migrate, as occurring at the 
end of cleavage in Clione. 

PULMONATA. 


Warneck (’50, pp. 131-135, 166-170) discusses the recurrent cleavage 
cavity in Limax and Lymneus. He describes its appearance soon after 
the two cells begin to flatten against each other ; also its growth and 
subsequent disappearance when the second cleavage plane appears. A 
similar phenomenon occurs at each succeeding phase of cleavage till the 
blastula stage is reached. He expresses the opinion that this “ heller 
Raum,” as he calls it, is a receptacle for albumen, and describes the 
expulsion of its albumen-like contents into the surrounding albumen at 
the time of the disappearance of the cavity. He explains the phenome- 
non as due to the acceleration of end- and ex-osmosis, attendant upon the 
greater activity of the nutritive and excretory functions of the cells and 
the disappearance and reappearance of the nucleus during the successive 
phases of cleavage, and correlates this activity of the cells with the 
origin of the ovum from a glandular tissue. Ganin (’73) mentions the 
relatively small cavity in Lymnzeus, and the larger cavity in Physa. 
In Helix, von Jhering (’75) finds a central cavity in the two-cell stage. 
The later stages of cleavage were not carefully followed by him, and no 
further mention of the cavity occurs in his work. 

Fol (’80, pp. 115 and 116) says: “ Pendant le travail du fractionne- 
ment, les sphérules prennent un aspect foncé et une forme arrondie. 
Les noyaux ne sont plus visibles et la cavité de fractionnement se perd 
dans l’obscurcissement de l’ceuf. Pendant les temps de repos les noyaux 
reparaissent, les sphérules s’affaissent les unes sur les autres, la cavité 
de segmentation est de nouveau visible. Dans ces périodes de repos, la 


Si i i 


KOFOID: DEVELOPMENT OF LIMAX. 99 


limite entre les sphérules apparait comme une ligne d’épaisseur trés 
appréciable et dont la transparence contraste avec la teinte foncée des 
cellules; c'est la coupe. optique de la couche de sarcode qui régne sur 
toute la surface des sphérules. Mais en outre nous remarquons dans le 
plan de contact des cellules voisines une accumulation de liquide, fait 
déja constaté par Warneck. Ce liquide va sans doute remplir la cavité 
de fractionnement, mais une partie est aussi expulsée sous forme de 
gouttelettes qui sortent généralment au pole nutritif. J’ai observé une 
seule goutte chez les Hétéropodes et les Pulmonés aquatiques. . . . La 
cavité de segmentation, ou blastocéle, reste presque nulle pendant que 
Yinvagination se forme et ne commence @ croitre qu’apreés le rétrécisse- 
ment du blastopore.” 

The propriety of the use of the term blastoccel or segmentation cavity 
to designate the lacunar spaces of the mesenchyma of the gastrula, as 
well as the spaces between the primitive blastomeres, is questionable. 
To be sure the mesenchymatous lacune are derived from the blastoccel 
rather than from an enteroccel, in Limax at least ; but our choice of 
terms is not limited to blastoccel and enterocel, and it seems preferable 
to apply to those spaces without epithelial lining which lie between the 
ectoderm and entoderm, and are traversed by loose mesenchymatous 
cells and prolongations of the ectoderm cells, a term not already set 
apart for another use. Previous to the formation of these lacune, all 
the cells of the intermediate layer exist as a solid mass obliterating 
the-cleavage cavity. When, however, in Limax the cavity reappears, 
as in Figure 54, it is as.a distinct space bounded by germ layers. It 
seems better, therefore, to apply to the spaces mentioned in the sec- 
ond quotation from Fol (p. 116) the same term which is used elsewhere 
for mesenchymatous lacunz not lined by a distinct epithelial layer, 
viz. schizoccel. 

Rabl (’79, p. 568) notes the presence of a cleavage cavity in the 
twelve-cell stage of Planorbis, and suggests the possibility of its presence 
in the eight-cell stage. It attains its maximum size when the embryo 
consists of twenty-four cells. He mentions the flattening of the blasto- 
sphere which follows this maximum condition, but does not speak of an 
obliteration of the cleavage cavity accompanying it, and considers that 
the cavity is still present at the time the mesoderm cells sink below the 
surface. He makes no suggestion of its recurrent nature at any period 
of its existence. 

Brooks (’80, p. 80) mentions in Physa the “ presence of a lens-shaped 
segmentation cavity, which is enclosed peripherally by the union of the 


100 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


two primary segments. This cavity persists from this stage until the 
completion of segmentation.” He does not refer to its recurrent char- 
acter in the earlier stages of cleavage, nor to the elimination of its 
contents in whole or in part. 

Joyeux-Laffuie (82), in his work upon Oncidium, a marine form with 
pulmonate affinities, makes no reference to a cleavage cavity. 

From my own observations on Planorbis and Physa, I have no doubt 
that the recurrent segmentation cavity is found in these forms, as in 
Limax ; but it is not developed in so marked a degree. I wish in this 
connection to call attention to the fact that the enclosing capsules and 
albumen of these forms are less dense than those of Limax, and that 
they are deposited tm the water. In Planorbis, which has somewhat 
more yolk than Physa, the cavity does not attain so great a size as 
in Physa. 


I shall not enter into an extended discussion, or a review of the litera- 
ture of the cleavage cavity in other groups of animals, especially of 
marine forms. I shall refer mainly to those forms which, by reason 
of their conditions of development, might be expected to throw light on 
the significance of the cleavage cavity. 

In Spongilla, likewise a fresh water animal, Maas (’90) finds no trace 


of a cavity in the solid morula stage, though he admits that there is at: 


the four-cell stage the intimation of one, which later entirely disappears. 
According to Brauer (’92) a cleavage cavity appears in Hydra at the 
eight-cell stage, but he makes no reference toa subsequent disappearance 
of the cavity. 
ROTIFERA. 


Zacharias (’85) finds a cleavage cavity in the two-cell stage of Philodina 
roseola. He does not figure it in the later stages, but speaks of its 
general appearance in all the eggs whose early stages he had observed. 

Zelinka (91) does not figure any cleavage cavity in the development 
of Melicerta or Callidina. 


ANNULATA., 


I have found no reference to a recurrent cavity in the marine forms 
of this group. In forms with much yolk, as Nereis, there may be no 
cavity whatever (Wilson 793); but in forms whose division is nearly 
equal, as in Eupomatus, a cavity appears at an early stage and persists 
until gastrulation (Hatschek ’86). 

Whitman (78) describes a cavity in Clepsine, which appears very 


. ~ 


oo ... —_ = s-. 


KOFOID: DEVELOPMENT OF LIMAX. 101 


early at the place where the first three planes of division cross one 
another. Its early and later history is not given. No mention is made 
of any obliteration or recurrence of the cavity. He suggests that ‘ the 
blastoccel, whenever it appears, forms as a necessary result of the cleav- 
age process. . . . The explanation of the cleavage cavity depends upon 
the fact that the cells push each other apart in cleaving.” 

This explanation was doubtless suggested by the cleavage of Clepsine, 
where there is considerable difference in size between the micromeres 
and macromeres, and the less mobile yolk-laden cells adapt themselves 
less readily to the changes in cleavage than the protoplasmic blastomeres 
of the egg of Limax. The difficulty of applying this mechanical explana- 
tion of the cleavage cavity to the phenomena observed in Limax will be 
patent to all. The blastomeres, in this form at least, are exceedingly 
plastic bodies, adapting themselves either to the presence or absence of 
a cavity, upon which profound changes in their form depend. Further- 
more, the “pushing apart” of the cells in cleavage is often the occasion 
of the obliteration of a cavity rather than its formation ; for the cavity, 
in the early stages at least, is frequently at its maximum Just before 
cleavage, and is obliterated or reduced in size at its close. 

Wilson (89) finds in the four-cell stage of Lumbricus a cavity 
which he labels ‘‘segmentation cavity,” and of which he says, it “ dis- 
appears afterwards and cannot be identified with the true blastoccel,” 
which is described for the thirteen-cell and later stages. The eggs of 
L. foetidus, for which this ephemeral cavity is figured, have tough cap- 
sules and thick albumen, similar to that of Limax. 

Vejdovsky (88-92) describes a distinct cavity in the two-cell stage 
of Rhynchelmis, and refers to the occurrence of a similar cavity in later 
cleavage stages. It is evident, however, from his figures, that the cavity 
is not so prevalent as it is in Limax. A similar cavity occurs in the 
two-cell stage of Allolobophora. A very interesting phenomenon was 
also observed by him in the six-cell stage of Allurus tetraéder. In the 
cytoplasm of the micromeres of this stage, a number of large contorted 
canals appear, which resemble very much the canals found later in the lar- 
val excretory cells of the embryo. These canals are filled with a clear 
fluid and “ hangen offenbar zusammen.” The accumulation of fluid in the 
canals results in an increase in the volume of the micromeres, and an 
obscuring of both cell boundaries and nuclei. Finally, by a powerful con- 
traction the fluid contents are expelled and the micromeres assume their 
original form and size, the cell boundaries and nuclei again becoming dis- 
tinct. It is evident that the author regards these canals as intracellular 


102 - BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


structures. No sections of this stage are figured, and the relation of these 
canals to cell boundaries is not determined. The fact that the presence 
of the canals obscures the boundaries between the cells, and that these 
canals are in continuity, suggests the possibility that they may be inter- 
cellular and therefore merely an exaggerated form of the anastomosing 
intercellular spaces so common in Limax. Vejdovsky does not suggest 
their relationship to the cleavage cavity, neither indeed does he regard 
a space found in the two-cell stage as having anything to do with that 
cavity. His grounds for this view, and his explanation of the phenome- 
non, are as follows (p. 105): “ Die Héhle zwischen beiden Furchungs- 
kugeln ist als Ueberrest der Vorgiinge zu betrachten, die sich bei der 
Bildung der Zellmembranen beider Furchungskugeln abgespielt haben. 
Diese Hohle zwischen den ersten 2 Furchungskugeln ist bereits oft beo- 
bachtet und als eine primiére Furchungshohle (!) gedeutet worden. Es 
ist tiberfliissig eine solche Auffassung zuritckzuweisen, einmal, dass es un~ 
moglich ist, dass eine Furchungshoble bereits zwischen zwei ganz gleich 
gestalteten Furchungskugeln zum Vorschein kommen k6nnte, ein ander- 
esmal, dass derartige Hohle Ofters auch wihrend des spiteren Furch- 
ungsprocesses zwischen je zwei Kugeln zum Vorschein kommt (vergl. 
Taf. IX. Fig. 11, 14). Gewiss ist diese Erscheinung von den Verhilt- 
nissen der Zell- und Kern-platte abhingig.” 

In the absence of the evidence upon which these opinions rest, it 
seems superfluous to discuss them. His suggestion that the formation 
of the cavity of the two-cell stage is dependent upon the phenomena 
of the division resulting in that stage is certainly not sustained by the 
facts. If his opinion were the correct one, we should find a similar 
cavity in the two-cell stages of all forms, fresh-water and marine alike. 


The preceding review of the literature shows that Warneck (50), 
Rabl (’79), Fol (80), and Brooks (80) have all noted the recurrent 
character of a cavity in the early stages of cleavage in the Pulmonates, 
but the three later writers have added little to the admirable observa- 
tions of the first named investigator. 

A glance at the summary of the literature on Prosobranch develop- 
ment shows an entire absence of any reference to a recurrent segmen- 
tation cavity in the marine forms, unless an exception be made with 
regard to McMurrich’s observations on the cavity in Fulgur. Whena 
cleavage cavity does occur, it appears at a very late stage in the seg- 
mentation, is comparatively small, and is never recurrent. The cleavage 
of the fresh-water Prosobranchs has not been fully studied except in 


KOFOID: DEVELOPMENT OF LIMAX. 103 


Neritina, but the cleavage cavity does not seem to be prominent here. 
It is however well marked in Amnicola. 

Among the Lamellibranchs there is the same absence of reference to a 
cleavage cavity in marine forms, but its recurrent nature is noted in 
Unio and Anodonta, probably also in Cyclas. <A cavity also occurs in 
Dreissena, but we do not know that it is recurrent. It seems probable 
that the encapsuled eggs of the non-marine Cheetopods may also present 
cases of a recurrent cavity. 

So far, then, as the literature and my own observations go, it seems 
that this recurrent cavity is confined to eggs developing in fresh water 
or moist situations, and reaches its maximum manifestation in forms 
enclosed in thick encapsuled albumen, like Limax. In these outward 
conditions of environment probably lies the explanation of this phe- 
nomenon. ,It is probably correlated with the nutritive and excretory 
processes of the egg, especially the excretory, as Warneck long ago sug- 
gested, for we have no evidence that the embryo depends in the cleav- 
age stages upon the surrounding albumen for its nutriment. The latter 
serves mainly as a protective covering during these stages, though later 
it is certainly used as food by the growing embryo. The metabolic pro- 
cesses taking place in the protoplasm of the blastomeres may be attended 
by the endosmosis of water from the surrounding albumen and its sub- 
sequent exosmosis. That part which passes out from the cell along the 
facets of contact with the other cells or on its inner surface, when such 
exists, contributes toward the formation of a cleavage cavity in some 
of its varying manifestations. When however the egg, or its envelopes, 
is bathed by a changing medium, as is the case with fresh-water and 
marine forms, the cavity is reduced or is altogether wanting, it being 
probable that the changing medium facilitates the solution and removal 
of the waste products from the surface of the egg. This is especially 
true of marine forms where the presence of the salt in the bathing 
medium doubtless facilitates the solution of the albuminous matters, 
and it is in these forms that the cavity is never recurrent and rarely 
present during cleavage. The eggs of the land Pulmonates, lacking as 
they do the salt, or even a changing medium, have the further disadvan- 
tage of a coating of dense albumen. This and its thick envelopes must 
necessarily impede the processes of excretion and respiration, a hin- 
drance which may be in part removed by the increase of the osmotic 
surface attendant upon the formation of the cleavage cavity, and also by 
the forcible removal of the products of excretion by the expulsion of 
the contents of the cavity. That there exists a physiological necessity 


104 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY, 


for some such process is evidenced by the enormous development of 
larval excretory and respiratory organs in these encapsuled embryos ; 
for it is in these forms that the primitive kidneys reach a maximum 
development, and the pulsating head vesicle and pedal sinus (podocyst 
or amnion of Jourdain) are found. 

A large amount of yolk in forms subjected to a fresh-water environ- 
ment, as Neritina, may conduce to the suppression of this ephemeral 
recurrent cavity. Its action may be purely mechanical, or it may per- 
haps be physiological, in that the process of cleavage, and therefore 
that of metabolism, take place less rapidly in such forms than in cases 
where there is less yolk and approximately equal cleavage prevails. 

In conclusion, then, it may be said that the existence of a cleavage 
cavity is dependent more upon the physiological necessities of the egg 
than upon the internal processes of cell division, or the mechanical neces- 
sities of cleavage, aud that its significance is preeminently physiological 
rather than morphological. 


3. Experimental. 


Fortunately the effect of salt in the surrounding medium is a matter 
that can be tested by actual experiment. As the eggs of Limax must be 
placed for examination in water, —an unnatural environment, — it has 
seemed best not to employ these, inasmuch as the water itself introduces 
a disturbing factor. Water very readily penetrates the membranes of 
the egg of Limax, and the albumen absorbs it so that the eggs become 
quite turgid. Accordingly, recourse was had to the eggs of fresh-water 
forms where it is not necessary to remove the eggs from their natural 
environment for experimentation. For this purpose the eggs of Amnic- 
ola limosa and Physa heterostropha were used. An egg mass of Physa 
showing the early stages of cleavage was divided into two equal parts, 
one of which was kept in the normal water of the aquarium while 
the other was placed in a salt solution made from the same water. 
The eggs were then kept under continuous observation, the phases of 
cleavage, and the size of the cleavage cavity in units of the ocular 
micrometer were recorded for both lots of eggs. There is some variation 
in individual eggs as to the rate of development, so that the observation 
of a number of eggs is desirable, in order that the predominant condition 
may be taken as the typical one. 

The eggs of Physa in the four-cell stage with a maximum cavity were 
placed in 0.75% salt solution. The elimination of the contents of the 
cavity occurred very soon afterwards, and slightly earlier in the eggs in 


- 


KOFOID: DEVELOPMENT OF LIMAX. 105 


the salt solution than in those under normal conditions. These later 
proceeded to cleave normaliy, forming a new cavity after the cleavage, 
which followed close on the obliteration of the cavity. The eggs in the 
salt water did not divide; nor did a central cavity reappear, small len- 
ticular spaces only being formed. All further activity seemed to be sus- 
pended during the four hours that the eggs were kept under observation. 
A portion of this lot was removed to fresh water after an exposure to 
the salt solution for an hour and forty minutes. In the course of an 
hour more cleavage took place, the cells passed through the rounded 
and flattened conditions, but during the two hours in which the eggs 
were observed no cavity appeared. These eggs seemed to be in a normal 
condition on the following morning. Cleavage had not advanced in the 
least in the eggs which remained in the 0.75% solution over night. 

The result of this experiment seems to show that the sudden transfer 
of the eggs to the 0.75% salt solution is inimical to their further devel- 
opment, and that it may have some influence in retarding the appearance 
of the cleavage cavity when they are restored to fresh water. 

A trial of 0.38% salt solution gave approximately the same results. 

When, however, the eggs of both Physa and Amnicola were placed in 
a 0.19% or 0.10% solution, cleavage was at first slightly delayed, occur- 
ring from five to fifteen minutes later than in the control eggs in the 
fresh water. After this initial delay, the cleavage seemed to progress in 
both lots with equal rapidity. With regard to the cleavage cavity, 
however, the two lots of eggs presented decidedly different results. 
When the transfer was made to the salt solution a maximum cavity was 
already present in both lots. Elimination occurred approximately at 
the same time in both, but the subsequent reappearance of the cavity in 
the eggs in the salt solution was delayed from fifteen to thirty minutes, 
and it never reached the size of that in the eggs in the fresh water. 
For example, the cavity in the control eggs attained a diameter of five 
to seven units, while that of the eggs in the salt solution was only three 
to four at the time of elimination. There were, however, a very few 
cases in which the cavity reached a diameter of five units. 

These somewhat limited experiments seem to accord very well with 
the observations on the cleavage cavity in fresh-water and marine forms, 
and with the interpretation given to the cleavage cavity in the preceding 
pages. They are also interesting in the light of Gruber’s (89) experi- 
ments upon fresh- and salt-water, Protozoa. When a species common to 
both environments, as Actinophrys sol, is transferred from salt to fresh 
water, there is a marked increase in the vacuolation of the protoplasm. 


106 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


This same vacuolation is characteristic of the fresh-water forms, as con- 
trasted with forms having a marine habitat. The reverse experiment 
has an opposite result, —a reduction of the vacuolation in the individ- 
ual when gradually transferred to salt water. Strictly marine forms, as 
Amoeba crystalligera, also show a marked development of vacuolation 
when they are brought into fresh water. These phenomena seem to 
show an increased activity of the protoplasm in the absorption of water, 
anda corresponding increase in the excretory function is also indicated 
by the much greater activity of the contractile vacuole in fresh-water 
as compared with marine forms. The physiological action of the recur- 
rent cleavage cavity of the Pulmonates is strikingly suggestive of the 
contractile vacuole of the Protozoa. The morphological distinction, that 
the one is intracellular while the other is intercellular, militates however 
against the homology of the cleavage cavity and contractile vacuole. 


VI. BLASTOPORE AND GASTRULATION. 


I have already called attention in the preceding pages to the changes 
in form characteristic of the stages of cleavage there discussed. The 
same causes produce similar changes in the later stages. We encounter 
rounded embryos with a central cavity (Plate VII. Fig. 47), and also 
much flattened individuals (Plate VII. Fig. 50). The latter are more 
common, and very generally present a more or less quadrangular outline, 
the sides of which are parallel] to the first two planes of cleavage, i. e. to 
the antero-posterior and transverse axes of the embryo ; the two or four 
mesoderm cells are symmetrically placed adjacent to the posterior side. 

In the case of two embryos, not figured, the mesoderm occupies a 
different position with reference to these four sides. Instead of lying 
adjacent to one of the sides, it lies in one angle, a position suggestive of 
the condition found in the forty-four-cell stage of Figures 39 and 40 
(Plate VI.), where the primary mesoblast does not seem to have under- 
gone a shifting into the median plane of the egg. In these cases it 
would seem to be necessary to orient the egg after the manner of Bloch- 
mann (’81) and Rabl (’79), with the first two cleavage planes cutting 
the axis of bilateral symmetry at an angle of 45°. 

It is in these flattened quadrangular embryos, containing from one 
hundred to one hundred and twenty cells, six or eight of which are 
mesoderm, that the first traces of gastrulation occur. Previous to this 
epoch the ventral face, i. e. vegetative pole, of the embryo has a much 


KOFOID: DEVELOPMENT OF LIMAX. 107 


more rounded contour than the animal pole. The blastoporic invagi- 
nation now appears as a broad shallow depression, involving a large 
part of the median surface of the vegetative pole. It is deeper at its 
rounded anterior margin and gradually diminishes in depth posteriorly. 
Figures 43 and 44 (Plate VII.) give the appearance of the depression as 
seen from the surface at this stage. Figure 48 (Plate VII.) is a sagittal 
section through the lateral margin of the depression, and Figure 49 is a 
median sagittal section, the anterior end being at the left in both figures. 
Figures 45 and 46 (Plate VII.) represent respectively the posterior and 
anterior regions of the blastoporic tract in transverse section. 

The dorsal surface also frequently shows a slight depression at this 
period. This may be due to the temporary obliteration or reduction at 
this time of the cleavage cavity, which is bounded laterally by the two 
bands of mesoderm (Plate VIII. Figs. 46, 48-50). The posterior end 
of the embryo is now slightly broader than the anterior, Figure 44 
(Plate VIII.). The broad ventral depression continues to deepen at 
the anterior end, and becomes narrower throughout its whole length. 
This results in a flattened embryo with an elongated median depression 
deepest at its anterior end. 

Such flattened embryos with an elongated blastopore have been fig- 
ured by Lereboullet (’62) and Lankester (’74) for Lymnzeus, and by 
Rabl (’79) for Planorbis. Fol (’80), who does not figure this stage of 
the Pulmonate embryo, states that he has not found embryos so much 
flattened as those described by Lankester. The earlier writers upon 
Limax have passed over this stage in silence, though Lankester (’75, 
Plate IX. Figs. 21, 22) figures two gastrule of Limax agrestis, both of 
which appear to be of a later stage, showing considerable difference 
in size between the cells of the two outer layers. 

The anterior border of this deepening blastoporic trough becomes 
more abrupt, and the lateral borders more sharply marked out, while 
the depression of the posterior region is gradually obliterated. The 
most marked change that accompanies this growth of the invagination 
is the increase in size of the anterior end of the embryo. Viewed from 
the ventral (Fig. 44, Plate VII.) or dorsal surface, it has had, up to 
this time, a rounded quadrilateral outline, usually with the posterior end 
the larger ; but in the stages represented in Figures 51-53 (Plate VIII.), 
the anterior end shows a marked increase in thickness as well as a 
lateral expansion. The thickening and lateral expansion of the anterior 
end are brought about by two influences, — the deepening invagination, 
and the accumulation of mesoderm in the antero-lateral region of the 


108 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


embryo. The blastoporic trough has now closed gradually from behind 
forward until there is a laterally compressed, cone-shaped pit directed 
antero-dorsally, and occupying a region at the vegetative pole directly 
opposite that where the polar globules are located, i. e. the blastopore 
at this period occupies a position corresponding to the centre of the 
vegetative half of the egg. 

The “landmarks” which assist in the orientation of this stage are : 
(1) The two lateral masses of mesoderm, now in continuity at the thick- 
ened region anterior to the blastopore, but separated posteriorly in the 
mid-dorsal region, where the ectoderm and entoderm retain their primi- 
tive contact. (2) The posterior region of the mesodermal bands, which 
marks the level of the posterior margin of the blastopore. These lie at 
the smaller end of the embryo. (3) The lateral projections, which may 
well be called ‘ velar” projections, for they occupy a position similar to 
that of the velum in Planorbis. The cells of this region are also some- 
what larger than those of the surrounding ectoderm (Plate VIII. Fig. 57). 
Owing to their constancy and prominence they are a valuable aid in the 
orientation of the embryo. 

At a stage succeeding this, represented in sagittal and transverse sec- 
tion respectively in Figures 56, 57 (Plate VIII.), a considerable trans- 
formation has been effected. The whole embryo has increased somewhat 
in size, owing to the growing vacuolation of the entoderm. The velar 
projections still persist and serve to emphasize the difference between 
the anterior and posterior ends of the embryo, as before ; but the rem- 
nant of the blastopore no longer occupies a median ventral position ; it 
is found at the posterior end of the embryo, Figure 55 (Plate VIII.). 
The relation of this to the preceding stage is a matter of considerable 
importance, owing to its bearing on the question of the fate of the 
blastopore. 

It is with considerable hesitancy that the consideration of this per- 
plexing question—the fate of the blastopore —is undertaken, for I 
cannot bring forward to support my conclusions the decisive evidence 
that can be offered in other forms, e. g. Umbrella. There are in Limax 
no definitely fixed and prominent landmarks, such for example as the 
anal cells of Umbrella. The germ layers themselves are not sharply 
differentiated from one another by the size of the cells, and the whole 
matter is further complicated by the existence of the recurrent cleavage 
cavity, the beginning of the vacuolation of the entoderm, and the 
development of the head vesicle, all of which contribute to profound 
changes in the external form of the embryo. 


yes 


. 


KOFOID: DEVELOPMENT OF LIMAX. 109 


Such evidence as there is seems to lead to the conclusion, that the 
orifice at this later stage, though posterior in position, is derived from 
the mid-ventral opening — the anterior end of the blastopore of an earlier 
stage — by a backward overgrowth of the anterior and lateral margins 
of that opening, the posterior lip of the blastopore being regarded as 
fixed, so that the remnant of the blastopore comes to occupy a position 
corresponding to the posterior lip of the blastopore of the earlier of these 
two epochs. If this is the case, we should expect to find intermediate 
stages showing steps in this overgrowth. The earlier stage, Figure 51. 
(Plate VIII.), shows some trace of it, for the pit of invagination has an 
antero-dorsal direction, i. e. in a ventral view the anterior lip of the 
blastopore somewhat overhangs the deeper portion of the invaginated 
layer, whereas the posterior margin rises obliquely to the level of the 
ectoderm. This overgrowth is accompanied by an accumulation of 
mesoderm in the anterior region. Although I have examined hundreds 
of embryos of about this stage, many of them killed especially for the 
determination of this question, very few good illustrations of this over- 
growth have been observed. Figure 53 (Plate VIII.) is a postero- 
ventral view of such a stage, showing the thickened anterior end with 
its velar projections. Occupying the mid-ventral region is an elevation 
which overhangs the site of the mid-ventral blastopore, whose posterior 
region is now marked by a trough-like depression. Figure 54 (Plate 
VIII.) is a nearly sagittal section, slightly oblique, through another egg 
of such a stage, showing the overhanging anterior lip and its contained 
mesoderm cells. Owing to the obliquity of the section the contact of 
the ectoderm and entoderm in the mid-dorsal region is not shown in this 
section. That such an overgrowth as I have suggested takes place is 
also shown by the conditions found in the later stage itself. 

Figures 56 and 57 (Plate VIII.) show that the dorsal wall of the archen- 
teron is much more vacuolated than the more recently formed ventral 
wall. They also show that there has been an accumulation of the meso- 
derm in this ventral region, and that the cells of the ectoderm are 
smaller in the ventral than in the dorsal half of the embryo. All of 
these facts seem to point to a more rapid growth in this mid-ventral 
part of the embryo. 

The conditions in the mid-dorsal region are of considerable interest. 
Assuming that the surface of contact between ectoderm and entoderm is 
constant now, as it has been during gastrulation, except when it is inter- 
rupted by the ephemeral cleavage cavity, we find that it no longer occu- 
pies the mid-dorsal region, but is shifted about 45° toward the anterior 


110 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


end of the embryo. This shifting is similar to that described by 


Heymons (93) for the velar field of Umbrella, and is perhaps homolo- 
gous with the shifting of the troch in the trochophore larva. 

A prominent funnel-shaped opening leads into the archenteron ; in 
the latest stages I have examined it still persists. This is the region 
where the anus is ultimately found, and Iam therefore inclined to the 
view that this remnant of the posterior part of the blastopore becomes 
the anus of the adult ; it certainly lies in the anal region. 

The mouth is formed at a later period by an invagination of the ecto- 
derm on the ventral surface of the embryo. Figure 55 (Plate VIII.) 
shows an embryo of a stage where the posterior opening leads into the 
archenteron, and the development of the head vesicle has obliterated all 
traces of the velar prominences. It will be seen that the ventral lip of 
the opening is prolonged posteriorly. This is the pedal elongation, which 
later becomes the podocyst. The shell gland arises on the dorsal sur- 
face as an invagination, posterior to the level of the velar region. The 
enlarged cells premonitory of the invagination are seen in Figure 57 
(Plate VIII.). 

The conclusions to which I have arrived as to the fate of the blasto- 
pore in Limax agrestis are directly contradictory to those of Fol upon 
L. maximus. According to him the blastopore becomes the permanent 
mouth. He seems, however, to have entirely overlooked the early stages 
in the history of the blastopore. His Figures 1 and 2 (Plate XVII.- 
XVIII.) represent stages in which the overgrowth of the blastopore has 
already taken place. The polar globules in his Figure 1 have also, it 
seems, been shifted somewhat, for they lie at the anterior end of the 
embryo. Furthermore, it is impossible to reconcile his Figures 3 and 4 
with his later stages, e. g. Figure 7. It seems much more probable 
that what he calls the mouth in Figures 3 and 4 is the anus, and that 
his shell gland is in reality the oral invagination. 

In concluding that the anus is formed from this persisting posterior 
part of the blastopore (actually or potentially), my conclusions agree 
with those of Biitschli (77), Lankester ?76), and Blochmann (’83) upon 
Paludina. Although the anus can thus be referred to the posterior 
region of the blastopore, it is not so certain, owing to the peculiar man- 
ner in which the anus is formed, that the mouth corresponds to the 
anterior part. It seems probable that in this case the mouth must be 
regarded as an entirely new structure. 


CAMBRIDGE, Mass., May 1, 1894. 


rd 


KOFOID: DEVELOPMENT OF LIMAX. AUS 


ADDENDUM. 


Since the completion of the preceding pages, Crampton’s paper on 
Reversal of Cleavage in a Sinistral Gasteropod (Annals N. Y. Acad. 
Sci., Vol. VIII. pp. 167-169, Plate V., March, 1895) has been received. 
Crampton has followed the cleavage of Physa heterostropha through 
the twenty-four-cell stage, and a reversal of the direction of the spiral 
is indicated for every cleavage. 


Ann Arpor, Micu., June 24, 1895. 


aby BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


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a ae 


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KOFOID: DEVELOPMENT OF LIMAX. 17 


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118 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


EXPLANATION OF THE PLATES. 


All figures were drawn from the eggs of Agriolimax agrestis, L., and are from 
preparations unless otherwise stated. A camera lucida was in every case employed. 
A, b, C, D, denote the first four cleavage spheres, A and B corresponding to 
the anterior quadrants, 6 and C to the right quadrants of the embryo. For the 
meaning of a, b, c, d, with their exponents, consult the explanation of the system 
of nomenclature of cells to be found on pages 40-43. 
The first, second, and third cleavage furrows are indicated by the Roman 


numerals I., II., I1I., respectively. 


Arrows are used to show the common origin of the cells thus connected, the 
head of the arrow occupying the cell nearer the animal pole of the egg. 


ABBREVIATIONS. 


arch. Archenteron. 

ast’cal. Astroceel. 

bl’ po. Blastopore. 

bl’po.a. Anterior end of blastopore. 

b?’po. p. Posterior end of blastopore. 

cav.sg. Cleavage (or segmentation) 
cavity. 

ench, Cells destined to form the 
shell gland. 


glb. pol. 
pd. 

pr’c, ame. 
pr’). vel. 
spa. vel. 
spa. Ins. 
vac. 

vS. Ce. 


Polar globule. 
Foot. 

Ameeboid process. 
Velar projections. 
Intercellular space. 
Lenticular space. 
Vacuole. 

Head vesicle. 


Koro. — Development of Limax. 


Fig. 
Fig. 
Fig. 
Fig. 
Fig. 
Fig. 
Fig. 


Fig. 


Fig. 
Fig. 
Fig. 
Fig. 


> 


Fig. 


Fig. 


ACD By ae 


Figures 1 to 7 drawn from the same living egg. X 275. 


Two-cell stage, beginning of flattened condition, 11.52 a. m. 

Same egg at 12.45 p.m. First trace of cleavage cavity. 

Same egg at 12.50 Pp. M. 

Same egg at 1.01 p. m. 

Same egg at 1.20 p.m. Maximum size of cleavage cavity. 

First (partial) expulsion of contents of cleavage cavity. 1.80 P. m. 

Same egg at 1.55 p. m. Spindles of the third generation present; the 
asters of the near ends of the spindles in focus. 


ae Crs reco) Ron rt 


Figures 8 to 13 drawn from another living egg of the same laying as 
Figures 1 to7. X 275. 


8. At 3.45 p. m. Division into four cells completed. Typical alternate 
arrangement of the four blastomeres. Viewed in the direction of the 
second plane of cleavage, therefore perpendicular to the direction of 
Figures 1 to 7. 

9. Same egg at 3.55 p.m. Formation of a cleavage cavity. 

10. Same egg at 4.05 Pp. M. 
11. Maximum development of the cleavage cavity. 4.35 P.M. 

12. Same egg after a gradual total expulsion of the contents of the cavity. 
4.45 p.m. Spindles of the fourth generation present. 

13. Formation of the eight cells of the fourth generation. Persistence of the 
cleavage cavity. 5.45 Pp. mM. 

14. Lateral view of two-cell stage. First cleavage plane just completed. 
Astroceels present. X 490. 


PEAWE T 


B Meisel, lith. Boston. 


i 


DEVELOPMENT OF Liva 


C.A.K. del. 


i is + ever. 
ry aie tl Ps ey ey yee ee 
gs way é 
) 


A : : 2 


wa lubace re eh | 
Ae Weak 2 ea a 


Koro. — Development of Limax. 


Fig. 15. 


Fig. 16. 
Fig. 17. 


Fig. 18. 
Fig. 19. 


PL 


All figures magnified 490 diameters. 


Two-cell stage from animal pole. 


ATE II. 


Spindles of the third generation pres- 


ent. No cleavage cavity. Deeper ends of the spindles (asters of B 
and D) shown by lighter lines. 


Four-cell stage from the animal pole. 


Second cleavage furrow almost 


completed. Cleavage cavity and lenticular spaces present. 
Four-cell stage from the animal pole. Cleavage cavity present. Spindles 


of the fourth generation. 


Same egg from the anterior end. 
Eight-cell stage from the animal pole, composed of the two quartets of 


the fourth generation, at1-d*! and at?—-d#2, 


plane just completed. 


Astroceels present. 


Third cleavage 


a 


rl kaplan 


MENT OF 


aD 
WO wt 


orD.- DEVEL 


ade KOF 


E Meigel Jith Boston. 


C.A.K. del. 


Korow. — Development of Limax. 


Fig. 20. 
Fig. 21. 
Fig. 22. 
Fig. 23. 
Fig. 24. 


Fig. 25. 
Fig. 26. 


PLATE III. 


All figures magnified 490 diameters. 


Eight-cell stage from the anterior end. Cleavage cavity and lenticular 
spaces present. Spindles of the fifth generation. 

View of the same egg from the animal pole. 

Sixteen-cell stage viewed somewhat obliquely from the right anterior 
quadrant, composed of the following cells: a>1-d51; a5-2—-d*?; 
a>3 — d>3; and a®*#- d®4. Cleavage cavity present. 

Lateral view of twenty-four-cell stage. Recent division of quartets 
resulting in 6.1, 6.2, 6.8, 6.4. Vacuolation at the animal pole. 

Oblique section of same egg. Fifth section in a series of twelve. Inter- 
cellular spaces at the animal pole. Cleavage cavity present. 
x, longitudinal section of intercellular space. 

Seventh section in same series. 

Transverse section of an embryo of about one hundred cells, showing 
vacuolated condition of all three germ layers. 


ee ery wwe 


pe tess 


Kororp.- DEVELOPMENT: oF Livax. 


BMeisel Jith.Boston. 


Koro. — Development of Limax. 


PLATE IV. 


All figures magnified 490 diameters. 


Figs. 27-80. Twenty-four-cell stage, composed of the following cells: a5-4—d54; 


Fig. 27. 
Fig. 28. 
Fig. 29. 


Fig. 30. 


a8 — 53; git — dit; gi8— (6-3, qb-2— q6-2; q6-1— 6-1, 

From the vegetative pole. 

From the animal pole, showing vacuolation. 

View of the right posterior quadrant (c). Vacuolation of the animal 
pole. 

Optical section along vertical axis cutting the quadrants b and d. 


Figs. 31,32. Twenty-four-cell stage, similar to the egg shown in Figures 27-80. 


Fig. 31. 
Fig. 32. 


Division of the quartet 6.2. 
View of the anterior end. Spindle in 4®?. Polar globule present. 
Same egg from the vegetative pole. Spindles in b%2, c6-2, q®2, 


—— = - 


B Meicel, lith Boston. 


> a 
AX. 


Koror.- DEVELOPMENT oF LIVA 
\.----- glib pol 


C.A.K. del. 


sesnuscsesetiet eee 


Bits teh arenas 


Korom. — Development of Limax. 


PLATE V. 


Figs. 38, 834. Twenty-four-cell stage, composed of the following cells: a5+- d+; 
ad-3 — 5-3; qbet— qb-4; q63— q6-3, q6-2— 52; q6-1— qb-1, 

Fig. 33. View of the left posterior quadrant (d) from below.  X 490. 

Fig. 34. Optical section of egg shown in the preceding figure, and in the plane of 
the paper, showing the right anterior hemisphere from the inside. 
Large cleavage cavity present. Ameeboid process (pr’c. ame.) on the 
cell a®-2, x 490. 

Fig. 35. Twenty-eight-cell, stage composed of the following cells : — a5 and 
76-8 6-8 qi.8 : A ee cies 
=o a oe a3 53, ght qd-4, gi3— 6-3; gb-2, J6.2 oat 6-2: 

a®-1—q61, View from the animal pole. Division of the quartets 5.4 

and 6.2. xX 510. 


. p68 [4 
Fig. 36. Twenty-five-cell stage, composed of a5-4, aD cot, dit; 5-3 5-3; GS-4 — 


d5-4; 63 q6-3; qS.2—d'-2 5 g6-1_q6-1, View of the animal pole. Divis- 
ion of a®+—d>-4. Spindles in c3, ch4. XX 510. 


Kororp.- DEVELOPMENT oF Limax. PLATE V. 


, C.AK del, B Meisel lith Boston. 


1 
-_ C - 
a , é F . 
r o 
> ‘ . 7 - 
E ‘ 
- v cee 
f os " 
, “ = 
> 1 e } V 
- ae 7 * 
_ hod - ~ ’ 
er be U4 - 
; a - — 1 
= = 
7 7 7 PF : 
“ 
i = 
. . 7 . 
S - = : & - ; 5 
| : = . pS ~ 
= 7 La « - 
in i - - * om - 
' ~ 
—— Le > _ H F 5 ed r. 
1 4 7 = x 
— J ¥ 
: : 4 
- q —__ 
1S “ s 
. ‘ 
_ 
ne : 
. i > 
=~ - > 
ey, 4 
*. “"z, n r 
= v 
aw! De ; 
u =— te 
— * —— 
* 
_ 


Korow. — Development of Limax. 


PLATE VI. 


Figs. 37, 38. Forty-cell stage, composed of the following cells : — 
a8 — 6-8; gh 7—qb7; q66— qi-8; gh5— qi-5; git qb4; qS3—q53; gid 
dit; qi3—di8; qi2—-d2; qai—del, 

Fig. 37. View from the animal pole. X 490. 

Fig. 88. View from the vegetative pole. Division of d’-? about to take place. 
The first mesoderm cell (d7*2= MW) with a large nucleus lies beneath its 
sister cell, d™1, only a small portion of it (shaded very dark) coming to 
the surface. X 490. 

Figs. 39, 40. Forty-two-cell stage, composed of the following cells : — 


cae F wee ee GALS Ps rs 
EPS IES oo GeO gaat OU as aT 


d63, gilt _q_i-t, ql3_ qi3; qi2—di2; all— dtl, 

Fig. 89. View of the animal pole. Division of b° into b° and b®6, Vacuolation 
at animal pole. X 490. 

Fig. 40. View of the vegetative pole of the same egg. X 490. 

Figs. 41, 42. Forty-five-cell stage, composed of the following cells : — 


a®8— 6-8; gi-7— 3-7; qi6 — 5-6; g65— 5-5; gi8—di8; qi-T_di7; 6-3 


? 
7+6 
56-3, — G3, gid — qt; qi8—di3; qi2%—pl2; ai-t— dil, 
Fig 41. View of the animal pole. Recent divisions resulting in a*-5, a®®, and 75, 
c'6, Xx 490. 
Fig. 42. View of the vegetative pole of the same egg. X 490. 


Kororp.- DEVELOPMENT oF Limax. PLATE YI. 


C. ALK. del. B Meise! lith Boston 


Koror. — Development of Limax. 


PLATE VII. 


Fig. 48. Young gastrula, seen from the posterior end, showing the broad shallow 
blastopore. > 300. 

Fig. 44. The same, viewed from the ventral surface. X 300. 

Figs. 45-50. Sections of gastrula stage. Mesoderm cells shaded dark. 

Fig. 45. View of the posterior face of the ninth section from the posterior end in a 
series of nineteen transverse sections, showing broad posterior portion 
of the blastopore. X 350. 

Fig. 46. View of the posterior face of the fifteenth section in the same series, show- 
ing the deepened anterior end of the invagination. x 350. 

Fig. 47. View of the right face (animal pole uppermost) of the eighth section in 
a series of sixteen sagittal sections through a blastula containing eighty 
cells, showing mesodermal pole cell and cleavage cavity. X 880. 

Fig. 48. View of the /efi face (animal pole above) of the eighth section of a series 
of twenty sagittal sections of an early gastrula, at about the stage of 
Figure 43, showing cleavage cavity and the mesodermal strand of the 
left side. X 850. 

Fig. 49. View of the /e/t face of the tenth section of the same series, showing at the 
right the mesodermal pole cell of the left side. 580. 

Fig. 50. View of the /eft face of the eleventh section of a series of seventeen 
sagittal sections through an embryo of about the stage of Figure 43, 
showing the mesodermal strand of the left side. A. Anterior; P. Pos- 
terior.  X 3880. 


Koror.- DEVELOPMENT oF LIMAX. PLATE VIL. 


B Meisel lith.Boston 


C.A.K. del. 


Kororp, — Development of Limax. 


Fig. 51. 


Fig. 52. 


Fig. 58. 


Fig. 54. 


Fig. 55. 


Fig. 56. 


Fig. 57. 


PLATE VIII. 


View of the ventral surface of a gastrula, showing blastopore and velar 
projections. X 360. 

View of the anterior face of the eleventh section from the anterior end in a 
series of nineteen transverse sections of an embryo of the stage of 
Figure 51, showing blastopore and velar projections. 850. 

View of the posterior ventral surface of a gastrula somewhat more ad- 
vanced than that represented in Figure 51. X 3800. 

Norr.— A defect in the shading causes the floor of the median 
groove (b/’po.) to appear elevated into a ridge. There is no such ridge. 

View of the /eft face of the seventeenth section in a series of obliquely 
sagittal sections of an embryo of the stage of Figure 53, showing cleavage 
cavity at the posterior end of embryo. X 3860. 

View of the ventral surface of an embryo more advanced than that seen 
in Figure 58, showing development of the cephalic vesicle and ti:e foot. 
x 490. 

View of the right face of the tenth section of a series of twenty-three 
obliquely sagittal sections of an embryo with blastopore in the poste- 
rior position. XX 3850. 

View of the anterior face of the eighth section, from the anterior end, of a 
series of sixteen transverse sections, showing archenteron, the velar 
ridge and enlarged cells (cnch.) in the region of the future shell gland. 
x 300. 


Kororp.- DEVELOPMENT oF Limax. 


OE 


B Meisel, lith Boston. 


Bi 
t 


ae 
our 
és 
45 
; 
tf 
© 


Bulletin of the Museum of Comparative Zoology 
AT HARVARD COLLEGE. 
Vou. XXVIII. No. 3. 


» CENTRAL AMERICA TO: THE GALAPAGOS, TO. THE WEST ‘COAST 
- ~*~ OF MEXICO, AND IN THE GULF OF CALIFORNIA, IN CHARGE OF 
ALEXANDER: AGASSIZ, CARRIED ON BY THE U.S. FISH COMMIS- 
SION STEAMER “ALBATROSS,” DURING 1891, LIEUT. COMMANDER 
~ Z. L. TANNER, U.S. N., COMMANDING. 


VILL: 


BIRDS FROM COCOS AND MALPELO ISLANDS, WITH NOTES ON 
PETRELS OBTAINED AT SEA. 


By C. H. Townsenp. 


Witn Two Cotorep PLATEs. 


CAMBRIDGE, MASS., U.S. A.: 


PRINTED FOR THE MUSEUM. 
Jury, 1895. 


reetare yet = 


Bulletin of the Museum of Comparative Zoology 
AT HARVARD COLLEGE. 
Vou. XXVIFE. No. 3. 


REPORTS ON THE DREDGING OPERATIONS OFF THE WEST COAST OF 
CENTRAL AMERICA TO THE GALAPAGOS, TO THE WEST COAST 
OF MEXICO, AND IN THE GULF OF CALIFORNIA, IN CHARGE OF 
ALEXANDER AGASSIZ, CARRIED ON BY THE U.S. FISH COMMIS- 
SION STEAMER “ALBATROSS,” DURING 1891, LIEUT. COMMANDER 
Z. L. TANNER, U.S. N., COMMANDING. 


XVII. 


BIRDS FROM COCOS AND MALPELO ISLANDS, WITH NOTES ON 
PETRELS OBTAINED AT SEA. 


By C. H. Townsenp. 


{Published by Permission of MARSHALL MCDONALD, U. S. Fish Commissioner.] 


Witn Two CoLorep PLaAtTes. 


CAMBRIDGE, MASS., U.S. A.: 


PRINTED FOR THE MUSEUM. 
Juty, 1895. 


- 


No. 3.— Reports on the Dredging Operations off the West Coast 
of Central America to the Galapagos, to the West Coast of Mex- 
ico, and in the Gulf of California, in charge of ALEXANDER 
Acassiz, carried on by the U. S. Fish Commission Steamer 
“ Albatross,’ during 1891, Lieut. Commander Z. L. TANNER, 
U.S. N., Commanding. 


[Published by permission of Marsuatt McDonatp, U. S. Fish Commissioner. ] 


XVII. 


Birds from Cocos and Malpelo Islands, with Notes on Petrels obtained at 
Sea. By C. H. Townsenp. 


Previous to the time the “ Albatross” called at Cocos Island, on 
February 28, 1891, nothing was known of its birds further than that 
it was the home of a peculiar cuckoo (Coceyzus ferrugineus, Gould), a 
single specimen of which was obtained during the voyage of H. M.S. 
“Sulphur,” about the year 1840. 

Although the collection made by the “ Albatross”’ is a small one, but 
three additional genera (and species) of land birds having been found, 
it is interesting as showing ornithological relationship between Cocos 
Island and the Galapagos Archipelago. 

Cocos Island is about 275 miles distant from Costa Rica, in latitude 
5° 32! 57" N., longitude 87° 2/10’ W. It occupies a position nearly 
midway between the mainland and the islands of the Galapagos group, 
and with the exception of Malpelo Island, an inaccessible barren rock off 
the Gulf of Panama, is the only connecting point of land. Like the 
Galapagos Islands, it is of voleanic origin, and has received its peculiar 
animal and vegetable forms from the mainland. The American origin 
of the forms of life upon the Galapagos Islands was demonstrated by 
Darwin, who made researches there more than half a century ago. It 


appears from a study of the birds alone, that Cocos Island is similarly a 
VOL. XXVII. — NO. 3. 


14 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


satellite of America, with the added interest of being a stepping-stone 
to the group of islands beyond it, some of whose ornithological features 
it bears. 

Darwin, the first to study the birds of the Galapagos Islands, de- 
scribed remarkable variations among them, even those inhabiting the 
same island, that made it difficult to separate them specifically. New 
forms brought to light by recent explorations, particularly those of the 
“ Albatross,” have only served as links to connect the species still more 
intimately, so that upon the Galapagos Islands there exists the most 
remarkable grading together of species known to ornithology. This is 
especially noticeable in the group of finches, in distinguishing which 
arbitrary measurements are employed, some of the smaller forms closely 
approaching Certhidea, a genus of the Cerebide. Into the gap between 
these (Cactornis and Certhidea) Cocornis from Cocos Island seems to fit. 
The relationship of the Cocos Island flycatcher Nesotriccus is equally close 
to Hribates inhabiting the Galapagos. In view of these facts, it is to be 
regretted that our limited stay at Cocos Island did not permit of a more 
thorough search for birds, as it is possible that other species exist in the 
elevated central part of the island which we were unable to reach. 

The island is abont four miles long by three wide, its central part 
having an elevation of about 1,700 feet. It is everywhere covered with 
the densest forest. Cucoanut trees are found upon the higher slopes, 
and tree ferns abound in the ravines. No tropical forest could be more 
dense and tangled. The rainfall is doubtless great, as each ravine con- 
tains a dashing stream. It is a garden spot in comparison with the arid 
Galapagos Islands. 

I am indebted to the kindness of Mr. Ridgway, Curator of Birds in 
the National Museum, for much valuable information on the ornithology 
of the islands of this region. 


COCOS ISLAND. 


Dendroica aureola, Gourp. 


Sylvicola aureola, Gould, Zool. Voy. Beagle, Part III. p. 86. 
Dendreca aureola, Salv., Trans. Zod]. Soc. Lond, Vol. IX. Part IX. p. 478. 


The two specimens of this warbler secured are not distinguishable from the 
species (D. aureola) inhabiting the Galapagos. Only one other was seen. 
The species is more closely related to D. petechia from Jamaica than the species 
of the mainland. 


TOWNSEND: BIRDS FROM COCOS AND MALPELO ISLANDS. 123 


Cocornis,! gen. nov. 


Intermediate between Cactornis and Certhidea of the Galapagos Islands, but 
distinguished from both in having a decidedly curved bill. The commissure is 
without the pronounced angle of the former and the gentle curve of the latter. 
It is nearest Cactornis, which it resembles in feet, coloration, and size, differ- 
ing in these respects from Certhidea, which it resembles more in the slender 
character of its bill. 


Tyre Cocornis Agassizi, sp. nov. 


Specific characters similar to Cactarnis scandens, but with bill more slender 
and curved, and less rounded, the culmen having more of the character of a 
ridge. 

Hab. Cocos Island. 

Adult male (Type No. 131680, Cocos Isl., Feb. 28, 1891, C. H. T.). Uni- 
form sooty black, except on under tail coverts, which are tipped with buff. 
Bill dark brown, lower mandible lighter; legs and feet brownish black. 
Length (skin), 4.85 inches; wing, 2.60; tail, 1.80; culmen, .56; gonys, .35 ; 
bill from rictus, .60 ; tarsus, .80; middle toe, .50. 

Immature male ? (No. 131682). Sooty black, washed with olive-buff, espe- 
cially below and on under tail coverts. Length (skin), 4.70; wing, 2.60; tail, 
1.75 ; culmen, .56; gonys, .35; bill from rictus, .60; tarsus, .80; middle toe, .50. 

Adult female? (No. 131690). Above sooty black, but with the feathers 
extensively edged and tipped with tawny olive, especially on upper tail cov- 
erts, where the black is almost entirely obscured. Edging fainter anteriorly, 
leaving crown quite dark. Middle and greater wing coverts edgéd and tipped 
with russet, tail russet-tipped. Below olive-buff, with the black appearing as 
a central streak in each feather, except on belly and under tail coverts, which 
are almost entirely olive-buff. The coloration of the upper parts blends 
gradually on sides, into that of the lower parts. Quills and tail narrowly 
edged with russet. Bill pale with dark tip. Legs and feet black. Length 
(skin), 4.50 in. ; wing, 2.50; tail, 1.65; culmen, .50; gonys, .30; bill from 
rictus, .55 ; tarsus, .80; middle toe, .50. 

In a series of eleven specimens of this bird, six males are sooty black, two of 
them entirely so (including the type specimen). Two have the bill entirely 
black. The other dark males have the bill dark brown. Five specimens, two 
females and three young males, resemble the female described above, and have 
the bill pale. The young males are somewhat darker than the females, while 
the full series of males exhibits a regular gradation from the light color of the 
female to the very dark color of the adult male. This is the only land species 
that seems to be really common. It is finch-like in its habits, always actively 
flitting from branch to branch. 


1 Cocos = the cocoa palm from which the island derives its name ; dpyis = bird. 


124 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


This remarkable species is named for Professor Alexander Agassiz, who was 
in charge of the work of the ‘‘ Albatross” at the time Cocos Island was visited. 


Nesotriccus, gen. nov.! 


Allied to Eribates of the Galapagos Islands, but with bill relatively onger 
and more flattened. Culmen separating the nostrils as a prominent ridge. 
Gonys less than half the length of lower mandible, terminating in advanee of 
nostrils. Tail relatively shorter. 


Tyre Nesotriccus Ridgwayi, sp. nov. 


Specific characters. Distinguished from the allied Eribates magnirostris in 
having no trace of rufous on inner webs of tail feathers, and no ashiness of 
throat and breast. It is also smaller, with nostrils separated by a sharp ridge. 

Hab. Cocos Island. 

Adult male (Type No. 131691, Cocos Isl., Feb. 28, 1891, C. H. T.). 
Above olive, brightening to olive-buff on rump ; tips of middle and greater 
wing coverts creamy buff; wings and tail dusky, with narrow olive-buff 
edgings. Below olive, suffused with yellow, brighter on belly and under wing 
and tail coverts, darker on breast and sides of head and neck : throat pale buff. 
Bill dark brown, with posterior half of lower mandible pale yellow. Legs and 
feet dark brown. Length (skin), 5.25 in.; wing, 2.40; tail, 2.20; culmen, 
.55; gonys, .35 ; bill from rictus, .80; depth at base, .18 ; tarsus, .80; middle 
toe, .45. 

Only one specimen of this bird was obtained, and to the best of my recollec- 
tion only two or three others seen. They were observed among the tree-ferns 
in a deep ravine at Chatham Bay. The species is named for Mr. Robert Ridg- 
way, Curator of Birds in the U. 8. National Museum. 


Coccyzus ferrugineus, Goutp. (Nesococcyx, Cab.) 


Coccyzus ferrugineus, Gould, Proc. Zool. Soc., 1845, p. 104. Zool. Voy. Sulph., 
Birds, I. p. 46. 


Only two specimens of this bird were obtained, and not more than three or 
four others seen. As in the case of the warbler (Dendroica) its relationships 
are with species inhabiting the West Indies, rather than with the forms of the 
mainland. The genus was not known to the Galapagos Islands until the 
voyage of the “ Albatross,” in 1888, when two specimens of Coccyzus melano- 
coryphus Vieill., a mainland form, were secured on Chatham and Charles 
Islands. 


1 ynoos = island; Triccus=a genus of tyrant flycatchers. 


TOWNSEND: BIRDS FROM COCOS AND MALPELO ISLANDS. 125 


Anous stolidus, Liny. 


Sterna stolida, Linn., Syst. Nat., Vol. I. p. 227. 
Anous stolidus, Ridgw., Proc. U.S. N: M., Vol. XII. p. 116. 


Abundant; four specimens collected. This species was noticed as most 
numerous, flying among the branches of the trees in the forest. The speci- 
mens, although resembling A. galapagensis, Sharpe, are apparently referable to 
A. stolidus. 


Sula, sp. 


Abundant, not collected. 


MALPELO ISLAND.! 


Creagrus furcatus (N&xzovux). 


Larus furcatus (Néboux), Prev. et des Murs, Voy. Venus, V. Ois., p. 277. 
Creagrus furcatus, Salv., Trans. Zool. Soe. Lond., Vol. IX. Part IX. p. 506. 
Creagrus furcatus, Ridgw., Proc. U. S. N. M., Vol. XII. p. 117. 


I shot four specimens of this rare gull during our short stop at Malpelo 
Island on March Sth, and saw several others upon the cliffs. The species is 
doubtless abundant there, as sea birds of several species swarm about the 
inaccessible summit of the island. 

But three specimens of this bird were known prior to the voyage of the 
‘* Albatross” to the Galapagos Islands in 1888, when I procured two speci- 
mens at Dalrymple Rock, Chatham Island. Malpelo Island is a new locality 
for Creagrus furcatus, the other examples known having all come from the 
Galapagos, with the exception of one from Peru, and the original specimen 
procured during the voyage of the “ Venus,” 1836-39, attributed, doubtless 
erroneously, to Monterey, California. Unless contained inthe Baur and Adams 
collection, only nine specimens are known at the present time, although the 
bird was discovered more than fifty years ago. 


PETRELS OBTAINED AT SEA. 
Oceanodroma cryptoleucuera, Ripew. 


Three specimens obtained off Wenman Island, Galapagos, April 4. They 
were attracted on board by the electric lights used on deck while dredging at 
night. 


1 Malpelo Island is a volcanic rock in Lat. 3° 59’ 7” N.; Lon. 81° 84’ 27” W. 
It is less than a mile in greatest length, with a height of over 800 feet. It is inac- 
cessible and without vegetation other than a small patch of bushes. 


126 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


Oceanodroma melania, Bonar. 


This species from the west coast of Mexico has been described by Ridgway as 
O. Townsendi in the Proceedings of the National Museum, Vol. XVI. p. 687, 
but is probably referable to O. melania. 

Nine specimens. I obtained the first on March 28, 1889, off Guaymas, the 
others in 1891; one off Acapulco, April 12th, and the others off Guaymas, 
April 21st. 


Halocyptena microsoma, Cougs. 
Halocyptena microsoma, Cours, Proc. Phila. Acad., 1864, p. 78. 


A single individual obtained while the “ Albatross” was dredging off Aca- 
pulco, April 12th. This is the third specimen known. I procured the second 
in 1888, off Panama. The original was taken in 1861, off Cape St. Lucas. 


Procellaria tethys, Bonar. 


Four specimens : two off Chatham Island, Galapagos, March 28th, and two 
on March 24th, 400 miles east of the Galapagos. 


Puffinus tenebrosus, Petz. 


Three specimens: one off Chatham Island, March 28th, the others off 
Wenman Island, April 4th. 


B Meise! Jith Boston 


NESOTRICCUS RIDGWAY1I, Townsend 


Adult Male 


l 
I 


B Meisel, lith Boston 


COCORNIS AGASSIZI, Townsend 


Adult Male and Female 


= L = 
L 


Bulletin of the Museum of Comparative Zodlogy 
Se AT. HARVARD COLLEGE. 
Vou. XXVII. No. 4. 


ORTS ON THE DREDGING OPERATIONS OFF THE WEST COAST OF 
CENTRAL AMERICA TO THE GALAPAGOS, TO THE WEST COAST 
S OF MEXICO, AND IN THE GULF OF CALIFORNIA, IN CHARGE OF 
ALEXANDER AGASSIZ, CARRIED ON BY THE U.S. FISH COMMIS- 
SION STEAMER “ALBATROSS,’ DURING 1891, LIEUT. COMMANDER 


' 42.1. TANNER, U.S. N., COMMANDING. 
XVIII. 

3 DIE COMATULIDEN. 
a Von C. Hartrave. 


ee (Published by Permission of MARSHALL McDONALD, U. S. Fish Commissioner.] 


iad se ee re 
Deli Me, 2 it $e 


With Four PLATEs. 


CAMBRIDGE, MASS., U.S. A. : 


PRINTED FOR THE MUSEUM. 
; AucustT, 1895. 


us s Ag Ft 
Rin te aoe 
res 


Bulletin of the Museum of Comparative Zoology 
AT HARVARD COLLEGE. 
Vou. XXVIII. No. 4. 


REPORTS ON THE DREDGING OPERATIONS OFF THE WEST COAST OF 
j CENTRAL AMERICA TO THE GALAPAGOS, TO THE WEST COAST 
OF MEXICO, AND IN THE GULF OF CALIFORNIA, IN CHARGE OF 
ALEXANDER AGASSIZ, CARRIED ON BY THE U.S. FISH COMMIS- 
SION STEAMER “ALBATROSS,’ DURING 1891, LIEUT. COMMANDER 
Z. L. TANNER, U.S. N., COMMANDING. 


XVII. 
DIE COMATULIDEN. 


Von C. HarTLAvs. 


{Published by Permission of MARSHALL MoODONALD, U.S. Fish Commissioner. ] 


WitH Four PLATEs. 


CAMBRIDGE, MASS., U.S. A.: 


PRINTED FOR THE MUSEUM. 
Auveust, 1895. 


—————————————— 


No. 4.— Reports on the Dredging Operations off the West Coast 
of Central America to the Galapagos, to the West Coast of 
Mexico, and in the Gulf of California, in charge of ALEXANDER 
Agassiz, carried on by the U. S. Fish Commission Steamer 
“* Albatross,’ during 1891, Lieut.-Commander Z. L. TAnNer, 
U. S. N., Commanding. 


[Published by Permission of Marsuatyt McDonatp, U. S. Fish Commissioner. ] 


XVIII. 


Die Comatuliden. Von C. HARTLAUB. 


Die Ergebnisse der Albatross Expedition sind, wie bereits von 
Agassiz’ mitgetheilt wurde, auf dem Gebiete der Crinoiden auf- 
fallend spiirlicher Natur gewesen. Wiihrend der Dampfer von 
andern Echinodermen, wie vor Allem Echiniden, Asteriden und 
Holothurien reiches Material erbeutete, besteht der ganze Fang an 
Crinoiden aus nur 7 Antedon-Arten und dem interessanten Calamo- 
crinus Diomede, welchen Agassiz schon beschrieben hat.? Unter 
den 84 Dredge-Stationen der Reise sind nur 10 zu nennen, die iiber- 
haupt Crinoiden lieferten. Von diesen fallen 6 auf die erste Fahrt, 
von Panama nach Cocos Island, 3 auf die zweite (3 Stationen bei 
den Galapagos) und eine auf die dritte Fahrt (Sta. 3424 Las Tres 
Marias). Ansehnliche Mengen von Exemplaren ergaben nur Sta. 
3385, Golf von Panama (Antedon tanneri n. sp.), Sta. 3424, Las Tres 
Marias, und Sta. 3357, nicht weit von Cap Mariato Point, wo u. a. 
eine Anzahl Antedons aus der Basicurva-Gruppe (Antedon agassizit 
vp. sp.) und der erwihnte Calamocrinus erlangt wurden. 

Verglichen mit den Crinoiden Schitzen der Blake-Expedition auf 
der atlantischen Seite Central-Americas war dies negative Resultat 
auf der pacifischen gewiss sehr itberraschend; allein so klein die 
Zahl der mitgebrachten Arten auch ist, die Albatross-Expedition 

1 Agassiz Al., General Sketch of the Expedition of the “ Albatross”’ from February 


to May, 1891, in: Bull. Mus. Comp. Zool. Harvard College. Vol. XXIII. No. 1. 


2 Agassiz Al., Calamocrinus diomede in: Mem. Mus. Comp. Zool. Hary. Coll. 
Vol. XVII. No. 2. 
VOL. XXVII — NO. 4. 1 


130 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


hat doch unsre Kenntniss von der geographischen Verbreitung des 
Genus Antedon sehr bemerkenswerth bereichert. Es hat sich nim- 
lich herausgestellt, dass eine Artengruppe dieser Gattung, die man 
nach den bisher bekannten Vertretern als vorwiegend arctigsch und 
nahezu antarctisch ansah, dies keineswegs ist. Die Eschrichti 
Gruppe, als deren siidlichster Verbreitungspunct auf der nordl. 
Halbkugel der 48, Grad nordl. Br. angesehen wurde (Antedon eschrichti 
bet Halifax) und welche auf der stidlichen Hemisphere die Magelhan 
Strasse und die Heard Islands als Fundorte hat, ist nicht minder in 
den Tropen zu Hause. An Individuenzahl nimmt sie in der Aiba- 
tross-Sammlung sogar bei Weitem die erste Stelle ein, leider auch 
beztiglich der Schwierigkeit, welche mir die systematische Beur- 
theilung der beiden Formen gemacht hat, von denen ich nachstehend 
mit allem bei Crinoidenarten néthigen Vorbehalte eine als neu 
beschrieben werde. Der Systematiker fiir Comatuliden ist ja inso- 
fern schlimm daran, als er fiir die mithsame Begriindung neuer 
Arten selten durch das glickliche Gefihl voller Sicherheit belohnt 
wird. 


I. ARTEN MIT AMBULACRALER TAFELUNG. 
BASICURVA GRUPPE Carp. Chall. Rep. XXVLI. p. 99. 


10-armige Antedon Arten, deren Radialia und untere Armglieder abge- 
plattete Seiten haben, und deren Pinnula-Ambulacra meistens getdfelt sind ; 
die zwei diusseren Radialia sind gelenkig verbunden. 


Uebersicht und Verbreitung threr Arten: — 

Gesammtzahl der Arten, 21. 

Geringste Tiefe: 49 Faden, Arapura See. — (Antedon denticulata 
Carp.) 

Grosste Tiefe : — 1600 Faden, Siidsee. — ( Antedon bispinosa Carp.) 


In Tiefen unter und bis zu 500 Faden, 13 Arten. 
49 Faden, Arapura See. Antedon denticulata Carp. 
88-262 “ Carribean Sea. A: | 
Str. of Florida. | ciples Ca 
“< ftexilis Carp. 


longicirra Carp. 
parvipinna Carp. 
pusilla Carp. 


140 ©“ Ki Islands. ee 


HARTLAUB: COMATULIDEN. : lish 


270 Faden, Str. of Florida. Antedon brevipinna Pourt. 


345 “off Japan. “© latipinna Carp. 
so: »<® Pacific, off 
Pangloa Isl. : tuberosa Carp. 
420-550 “ S§. Atl. off Tristan da 
Cunha, Ascension. “  — multispina Carp. 
a aculeata Carp. 
500 <‘“ Meangis Isl. «© gracilis Carp. 
. valida Carp. 


In Tiefen von iiber 500-1000 Faden, 9 Arten. 


420-550 Faden, S. Atl. Tristan da 
Cunha, Ascension Axtedon multispina Carp. 


; tncerta Carp. 
630 ‘ Kermadecs. “©  echinata Carp. 

os basicurva Carp. 

630-1350 “ « und Fiji. oi breviradia Carp. 
610-630 “ oe ue incisa Carp. 

740 ‘ off Portugal. as lusitanica Carp. 
782 “ — off Mariato Point. ee agassizii, Hartl. 
950 “ Port Jackson. e spinicirra Carp. 


In Tiefen von iiber 1000 Faden, 3 Arten. 


1350 Faden, Fiji. Antedon acutiradia Carp. 
115 10 a al ‘- breviradia Carp. 
1600 “ Siidsee. le bispinosa Carp. 


Antedon agassizii n. sp. 
Taf. I. Fig. 4, 7,8; Taf. II. Fig. 16, 18, 19; Taf. III. Fig. 23; Taf. IV. Fig. 26. 


Centrodorsale von miissiger Grésse, kuppelférmig, am dorsalen 
Ende cirrusfrei und mit kleinen Dornen bedeckt; 15-22 dine, 
namentlich in der fusseren Halfte stark comprimirte Cirren von 
etwa 40 mm. Linge; dieselben stehen in 2 und stellenweise 3 Hori- 
zontalreihen und bilden bisweilen auch Verticalreihen. Grdsste 
Anzahl der Cirrusglieder etwas iiber 60; davon die beiden ersten 
kurz, das dritte linger, das 4. mal solang als das dritte, das 6. am 
lingsten und an langen Cirren 2} mm. messend; von ihm ab nimmt 
die Liinge der Cirrusglieder rasch ab bis circa zum 20. Gliede, auf 
welches eine Reihe kurzer, allmilig kleinerwerdenden Glieder 
folgen; im Verlaufe dieser letzteren ist die dorsale Kante des Cirrus 
deutlich gezackt. Manche Cirren haben an den letzten 30 Gliedern 
einen ausgesprochenen Dorn. Dorn des vorletzten Gliedes mitunter 
schwach. 


Los BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


Erste Radialia eben sichtbar; die zweiten kurz, seitlich voll- 
kommen frei, und fiir das rhombische Axillare, mit welchem sie 
einen schwachen Buckel bilden, nicht eingeschnitten. <Avillare 
rhombisch, distalwiirts stark verbreitert und hier zuweilen mit ver- 
tikal abgeplatteter Seite den Nachbaraxillarien angelagert. Zweites 
Radiale und Axillare sind mit kurzen stumpfen Dornen besetzt. 
Manchmal sind einzelne 2-gliedrige Distichalserien vorhanden. 

10-12 rundliche schlanke Arme vou betrichtlicher Linge und ganz 
rauher Oberfliche. Die Armglieder bis zur zweiten Syzygie tragen 
auf ihrem proximalen und distalen Rande kleine aufrechtstehende 
stumpfe Dornen-nach Art derer auf den Radialien. 

Erste Brachialia ziemlich kurz, in der hinteren Halfte ambulacral- 
wirts verbreitert und hier mit vertical abgeplatteter Seitenfliiche 
dem ersten Brachiale des Nachbarradius angelagert. Diese Seiten- 
fliiche bildet die Fortsetzung der des Axillare und somit eine Ge- 
sammtfliiche mit dieser.— Auf der Innenseite berithren sich die 
ersten Brachialia in ihrer proximalen Halfte. 

Zweite Brachialia etwas linger als die ersten und distalwirts ver- 
breitert. Sie sowohl als die 3. Brachialia stehen in enger Berithrung 
mit dem 2. und 3. Brachiale des Nachbararms; diese Berithrungs- 
stelle ist, da sie ganz ambulacralwirts liegt, schwer zu bemerken. 
Sie ist von der gleichen Contactfliche des ersten Brachiale durch 
eine Liicke getrennt.— Die auf das 3. folgenden 7 Glieder sind 
etwas breiter als lang und in der Mitte eingeschnirt. Vom etwa 
9. an nimmt die Liinge der Armglieder zu, und ihre Form beginnt 
mehr dreieckig zu werden, und so bleibt sie ungefihr vom 20. bis 
zum 30. Gliede. Vom etwa 23. Gliede an (2} mm.) nimmt die Linge 
wieder allmiilig ab. Auf die dreieckige Gliedform folgt eine mehr 
trapezoide, und in der zweiten Armhilfte erschienen die Glieder ditten- 
artig in einander gesteckt (Taf. II. Fig. 16). Die distalen Rander 
siimmtlicher Armglieder sind stark aufgeworfen und in der zweiten 
Armhilfte fein dornig. Hier gehen von ihnen Lingsricfen aus, 
welche iiber die distale Gliedhalfte hinziehen. 

Die Dicke der Arme nimmt ganz allmilig ab; die Armlainge 
betrigt bis 170 mm.; die Zahl der Armglieder bis zu 130. 

Erste Syzygie im 3. Brachiale; zweite vom 13.-15., dann in Ab- 
standen von 2-3 Gliedern. 

Die Pinnula des zweiten Brachiale (Taf. II. Fig. 18) ist ziemlich 
steif, 9-11 mm. lang und zihlt 16-18 kurze, glatte Gleder, von 
denen die ersten 4 — vor Allem aber die 2 ersten — breiter als lang 


HARTLAUB: COMATULIDEN. oo 


sind. Es ist diese Pinnula an ihrer Basis seitlich abgeflacht und breit 
und nach etwa dem 6. Gliede spitzt sie sich schnell und fein zu. Thre 
diusseren Gleder haben feine, bedornte Distalriinder; die Glieder 
der proximalen Halfte aber glatte ; jedoch sind bei ihnen die ambu- 
lacralen Kanten gezackt. Das 2.-5. Glied hat eine Art Kiel; die 
Verbindung der mehr basalen Glieder unter sich ist eine lockere und 
dies in erhéhtem Grade an der folgenden Pinnula, deren Basalglieder 
dementsprechend eine etwas andre Form haben; doch sind auch 
diese etwas abgeflacht. Es hat das aber schon bei den unteren Glie- 
dern der 3. Pinnula, die rundlicher ist, ein Ende. 

Die Pinnula des 4. Brachiale ist ktirzer und hat etwas weniger 
Glieder, auch ist sie weniger steif und bedeutend feiner gebaut; sie 
hat wie die folgenden Pinnulz vorspringende, feine bedornte distale 
Rander. 

Die dann kommenden Pinnulze werden bei etwas abnehmender 
Linge (geringstes Maass 6 mm.) noch zierlicher und ihre Glieder 
werden lainglicher. Vom 14. Brachiale nimmt die Linge der Pin 
nule wieder zu bis zu einem Maximum von 23 mm., und 22-23 
Gliedern. Diese letzteren werden bedeutend linger als breit mit 
Ausnahme der 2 ersten und besonders des Basalgledes, welches 
kurz und breit bleibt und eine trapezoide Form hat. 

Die Pinnula des 3. Brachiale ist etwa 2 mm. kiirzer als die des 
zweiten und entschieden schwiicher. 

Ambulacralfliiche der Arme und Genitalpinnule deutlich getiifelt. 
Die Pinnule haben Seitenplatten (Taf. 2, Fig. 19). 

Sacculi klein und wenig auffallend; mit den Seitenplatten alter- 
nirend. 

Scheibe stark eingeschnitten ; vollstiindig getifelt. 9 mm. Dm. 

Farbung in Spiritus gleichmassig griinlich gelb, zum gréssten 
Theil durch den stark ebenso gefirbten Alcohol ausgezogen. 

Klafterung bis 35 em. 

Fundorte Station No. 3357, nicht weit von Cap Mariato Point 
(Bucht von Panama), in 782 Faden, “‘ Modern Greensand”; Boden- 
temperatur 38,5 F.— Station No. 3408, Galapagos, 684 Faden, Glo- 
bigerinen Schlamm ; Bodentemperatur 39,5 F.— Station No. 3409, 
Galapagos, 327 Faden, Schwarzer Sand; Bodentemperatur 42,3 F. 

Bemerkungen : — 

Station No. 3408 lieferte ein kleines 1l-armiges Exemplar. Es 
ist dadurch ausgezeichnet, dass seine erste Pinnula weniger glatte 
Gliedrinder hat, als es die Regel ist. Sie sind fein bedornt, was bei 


134 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


den iibrigen Exemplaren nur an den dussersten Gliedern der betr. 
Pinnula der Fall ist. 

Station 3408 ergab zwei noch kleinere Exemplare (Armlinge 44 
em.) und 2 ganz winzige, bei denen das erste Radiale vollkommen 
sichtbar ist und stumpfe, kurze, aufrechtstehende Dornen besitzt. 

Bemerkenswerth fiir die neue Species ist neben dem Dimorphis- 
mus in der Anzahl der Arme, die Beschrenkung der wandartigen inter- 
radialen Contactfldchen auf das Axillare und erste Brachiale, und der 
Umstand, dass dieselben sogar vollkommen fehlen kénnen. Die 2. 
Radialha sind seitlich ganz frei; sie berithren sich nur an den Aus- 
senkanten ihres verbreiterten proximalen Endes. Zwischen dieser 
Beriihrungsstelle und dem Contact der Axillaria befindet sich ein 
offner Zwischenraum, ein Loch. Aehnlich scheint sich Antedon 
multispina Carp. zu verhalten, doch ist der Autor geneigt bei ihr 
dies Vorkommen als Jugendcharakter zu deuten. [‘ The 2 outer 
radials, especially the second, which are very short can hardly be 
described as wall-sided,” Chall. Rep. XX VI. p. 117.] 

Zu beachten ist sodann die Klafterung von Antedon agassizit, 
welche mit 35 em. die fast aller andern Arten der Gruppe weit tiber- 
trifft. Am nichsten kommt ihr Antedon valida Carp. mit “25 cm. 
spread.” Uebertroffen wird ihre Grésse durch Antedon flexilis (Basi- 
curva und Spinifera Gr.) mit “55 cm. spread” und Antedon robusta 
(Spinifera Gr.) “spread nearly 50 em.” 

Entschiedene Aehnlichkeit verbindet die Art durch die Bedornung 
ihrer Radiala and unteren Brachialia mit Antedon bispinosa, bei 
welcher die Kelchglieder und die untersten Brachiaha der Abbildung 
nach zu urtheilen (Chall. Rep. XXVI. Pl. 20) nicht nur an den 
Rindern sondern auf ibrer ganzen Oberflache mit kleinen stumpfen 
Dornen besetzt sind. Bei Antedon agasiizii geht indessen dieser 
Schmuck weiter den Arm hinauf als bei jener Art und die kleinen 
ganz aufrechtstehenden Dornen sind auf den proximalen und distalen 
Rand eines jeden Gliedes beschrankt, in der Weise, dass jede Glied- 
verbindung durch 2 Querreihen von Dornen gekennzeichnet ist. 

Beztiglich das Calyx verweise ich auf Tafel 1, Fig. 4, 7, 8. — Die 
Cirrusgruben sind ziemlich gross und besitzen einen ansehnlichen 
Gelenkkopf. Die ventrale Fliche des Centrodorsale ist annahernd 
pentagonal und die 5 Ecken ragen etwas vor. Die dorsale Flache 
des radialen Pentagons zeigt Spuren eines Basalsterns. Im Trichter 
erkennt man, etwas tiefer als der dorsale Rand gelegen, die soge- 
nannte Rosette. Die Gelenkflichen der Radialia (Fig. 8) lassen 


HARTLAUB: COMATULIDEN. 135 


eine Anzahl Gruben erkennen, von denen die zuniichst ventralgele- 
genen Muskelgruben sind. Sie haben anniihernd dieselbe Grdsse, 
wie die durch eine schrige Leiste von ihnen getrennten Ligament- 
gruben und besitzen eine glatte Oberfliche. 

Die ambulacrale Tafelung der Pinnula (Taf. 2, Fig. 19, Taf. 3, Fig. 
23) besteht aus Seiteriplatten und Deckschuppen. Die Deckschuppen 
sind ganzrandige, feinmaschige, annahernd ovale Blattchen; sie ruhen 
auf den Seitenplatten und sind wahrscheinlich beweglich, da ihre 
Stellung eine sehr wechselnde ist. Bald liegen sie dachziegelartig 
flach tibereinander, bald sind sie mehr aufgerichtet; betrachtet man 
sie in dieser Lage von unten, so sieht man, dass sie eine alternirend 
schriige Stellung zur Liingsaxe der Pinnula haben. Ihre genaue 
Befestigungsart zu bestimmen ist mir einstweilen nicht gelungen. 
Die Elemente sind fusserst klein, sehr zerbrechlich und schwer zu 
isoliren. Sie werden durch Weichtheile festgehalten, die selbst nach 
mehrere Minuten langen Kochen in Kalilauge nicht zerstért werden. 
Allzu langes Kochen in Kalilauge vertragen, die sehr diinnen zerbrech- 
lichen Plattchen andrerseits auch nicht. Beziiglich der Seitenplatten 
sei bemerkt, dass sie im Zickzack stehen. Sie bilden so auf jeder 
Seite der Ambulacralrinne eine Art spanischer Wand. In den 
Nischen mehr oder minder verborgen, legen die Sacculi. Die vor- 
springenden Ecken werden meist von 2 aneinander stossende Platten 
gebildet (Fig. 19 und 23 se, se’); an anderen Stellen, so namentlich 
am Ende einer Pinnula, macht es aber den Eindruck, als ob sie von der 
Abknickung oder Biegung ein und derselben Platten herriithrten. — 
Die Seitenplatten sind weitmaschiger als die Deckplatten, oft von 
anniihernd viereckiger Gestalt und ausgezackten Randern. An eini- 
gen Stellen sieht man (Taf. 2, Fig. 19), dass durch diese regelmissig 
wiederkehrenden Auszackungen 2 Liicken in der Beriihrung der 
Kanten entstehen. Die oberen dieser Liicken (Fig. 19 s.) werden 
durch die Saccuh ausgefiillt, durch die unteren (Fig. 19 t.) treten 
die Tentakel nach aussen. : 

Die beschriebene Species Antedon agassizii n. sp. ist, wie wir gese- 
hen haben, dimorphisch, d. h. sie kommt nicht nur in 10-armigen 
Exemplaren vor sondern auch nicht selten in solchen, die 11 oder 
12, ja vielleicht nochmehr Arme besitzen und diese Exemplare 
wiirden, da sie 2 Distichalia haben, zu einer ganz andern Serie der 
Carpenterschen Eintheilung gehoren, namlich in die Spinifera Gruppe. 
Da Antedon agassizit darin nicht vereinzelt dasteht, sondern innerhalb 
derselben Gruppen Antedon flexil’s, duplex, lusitanica sich ganz gleich 


136 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


verhalten, ebenso Antedon multispina, nur mit dem Unterschiede, 
dass sie auf Grund ihrer 5 Distichalia zam Theil in einer noch an- 
dern Serie figurirt, so wirft sich hier naturgemiiss die Frage auf, ob 
eine Eintheilung, die derartige Doppelplacirungen nothwendig 
machte, natiirlich und practisch war. Unserer Auffassung nach hat 
Carpenter in der Zusammenfassung der 10-armigen Antedons zu einer 
Serie einen entschiedenen Fehler gemacht. Er hat Arten ohne alle 
sonstige Gemeinschaft miteinander darin vereinigt wie z. B. die 
dusserst verschiedenen Arten der Basicurva und Tenella Gruppe, 
wihrend er andre die durch den Besitz einer ambulacralen Bewaff- 
nung, durch ihre horizontale und bathymetrische Verbreitung im 
Gegensatz zu den ibrigen Species stehen, weit voneinander trennte. 
Auch unter den Arten mit unbedeckten Ambulacren finden sich 
solche die bald 10-armig, bald mehrarmig erscheinen ; auch sie besti- 
tigen, dass der Character der 10-Armigkeit kein zuverlissiger ist 
und deshalb nicht hatte Veranlassung geben diirfen iibrigens soweit 
verschiedene Species in einer Serie systematisch zu verbinden. 
Jeden, der sich mit Comatuliden beschaftigt hat, muss der iiberein- 
stimmende Charakter im Habitus der Basicurva, Spinifera und 
Granulifera Gruppe frappirt haben, dazu kommt, dass sie viele 
Tiefseeformen aus dem pacifischen Ocean und dem Caraibischen 
Meere enthalten, wihrend die grosse Mehrzahl der iibrigen flacheren 
Gewiassern angehéren und indischen Ursprungs sind. (So vor Allem 
die zahlreichen Arten der Savignyi und Palmata Gruppe.’) |Unsre 
Ansicht geht also dahin jene 3 Gruppen und die Elegans Gruppe, bei 
welcher die tiusseren beiden Radialia im Gegensatz zu allen andern 
Antedon Arten durch Syzygie verbunden sind, bei welchen aber die 
Scheibe und die Ambulacralia innerhalb der aussersten Axillaria 
getiifelt sind, zu einer grossen Serie zu vereinigen und sie einer 
zweiten Serie gegeniiber zu stellen, welche die Gruppen mit unge- 
tifelten Ambulacren umfasst. Es stellt sich das iibersichtlich fol- 
gendermaassen dar. 


I. Arten mit getifelten Ambulacren: Serie I. 
a) Die 2 ausseren Radialia durch Gelenk verbunden. 


; Basicurva Gruppe. 
10 Arme Noten is 
2 Distichalia Spinifera  ¢° 
3 Distichalia Granulifera ‘¢ 


1) Hartlaub, A., Beitrag zur Kenntniss der Comatulidenfauna des Indischen Ar- 
chipels in: Nova Acta Leopold Car. Akad. Bd. LVIII. Nr. 1.1891. 4o. 


HARTLAUB: COMATULIDEN. 137 


b) Die 2 ausseren Radialia 
durch Syzygie verbunden  Elegans Gruppe. 


II. Arten mit ungetifelten Ambulacren: Serie II. 


Eschrichti Gruppe. 


10 Arme Milberti “ce 

Tenella ce 
2 Distichalia Palmata “ 
3 Distichalia Savignyi us 


II. ARTEN OHNE AMBULACRALE TAFELUNG. 


ESCHRICHTI GRUPPE Carp. Cuauu. Rep. XXVI. p. 136. 


“10 Arme. Die ersten zwei oder drei Pinnula Paare lang und 
geisselfOrmig, mit zahlreichen kurzen und breiten Gliedern.” 
Uebersicht und Verbreitung der Arten : — 
Zahl der bekannten Arten: 9. 
Bathymetrische Verbreitung, 20-782 Faden. 
In Tiefen unter und bis 200 Faden : 
20-632 Faden, Le Have Bank. Antedon Eschrichti Mill. 


25-410 “ Smith Sound. “  quadrata Carp. 
30 “ Magellan Str. “  magellanica Carp. 
75 “  Siidsee. “*  antarctica Carp. 
132 “ Kara See. “ barentsi Carp. 
P50 95% ** Sudsee. “australis Carp. 


In Tiefen iiber 200 Faden: 


20-632 Faden, Porcupine Sta. 57. Antedon eschrichti Carp. 
25-410 “ Davis Str. “<<  quadrata Carp. 
286 ‘ Bucht v. Panama. “ tanneri Harti. 
676-782 “ bei Mariato Point. “« — rhomboidea Carp. 


Die héchste bis jetzt fiir die Gruppe nachgewiesene Bodentempera- 
tur war 30,2 F. (= 1,67 Celsius) (A. australis zw. Heard Island und 
Kerguelen]. Auf Station 286 des Albatross aber—dem Fundort 
der neuen Art A. tannert betrug die Bodentemperatur 45,9 F. (= 
7,70 Celsius). 


138 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


Antedon rhomboidea Carp.’) 
Taf. I. Fig. 1, 2, 3, 6, 10, 11; Taf. II, Fig. 12, 14, 15,17; Taf. III. Fig. 24. 


Centrodorsale flach kuppelférmig, mit einer cirrusfreien Aushoh- 
lung im Scheitel; circa 60 Cirren in 4-5 Reihen. Cirren gleich- 
missig diinn, mit etwa 40 Gliedern, die vom 4. bis etwa 10. langge- 
streckt sind und bis gegen das 20. Glied noch linger wie breit 
bleiben. Die Cirren sind mit Ausnahme der 7 oder 8 untersten 
Glieder stark comprimirt. Von der dorsalen oder ventralen Kante 
betrachtet, erscheinen daher auch die dusseren Glieder noch lang- 
lich. Das dorsale Profil der distalen Halfte des Cirrus erscheint 
gezackt weil auf dieser Seite die Enden der Glieder vorspringen und 
selbst die Form kleiner Dornen annehmen, Die Linge der Cirren 
erreicht zuweilen 6 cm. 

Erste Radiale kurz; zweite sehr kurz, seitlich nicht in Beriihrung 
unter einander, auf ihrer Verbindung mit dem Axillare eine starke 
knopfartige Erhebung, die an jiingeren Exemplaren aber kaum ange- 
deutet ist. Axillare ziemlich gross, dreieckig; seine proximale 
Seite ist bedeutend breiter als die daran stossende distale Kante des 
2. Brachiale. 

10 Arme, die in ihrer proximalen Hilfte ziemlich dick und glatt 
sind und iiberall rundliche Glieder haben. Erste Brachialia kurz, 
auf der Innenseite mit einander nicht in Beriithrung; auf ihrer Ver- 
bindung mit dem zweiten ein starker Knopf; ahnliche knopfartige 
Erhebungen von schwicherer Entwickelung bemerkt man alternirend 
seitlich gelegen bis zum 10. oder 11. Gliede. Zweites Brachiale 
annihernd dreieckig. 5. Brachiale quadratisch (Syzygie) ; folgende 
Glieder bis zum 8. (2. Syzygie) von fast derselben Liinge aber mehr 
trapezformig. Auf das 8. folgen 2 oder 3 kirzere, scheibenformige 
Glieder und auf diese ausgesprochen dreieckige. In der éusseren 
Armhilfte, wo die Glieder feindornige, verdickte itiberragende Dis- 
talriinder bekommen, wird die Gliedform wieder mehr trapezoid. 

Erste Syzygie im 3. Brachiale, zweite im 8., dritte Syzygie im 13. 
oder 14. Gliede, seltner schon im 12. oder erst im 15. Dann in 
Zwischenriumen von 2-5 Gliedern. 

Erste Piunula (Br. 2) lang und diinn geisselférmig, mit etwa 50 
ausschliesslich kurzen Gliedern; 18 mm. lang; zweite etwas linger 
und von fast gleicher Form; ihre Glieder sind etwas dicker und 


1) Chall. Rep. XXVI. p. 148. Pl. XIL Fig. 1&2; Pl. XXIV. Fig. 1-3. 


HARTLAUB: COMATULIDEN. 139 


meist linglich. Dritte Pinnula kiirzer, mit viel weniger, aber be- 
deutend verliingerten Gliedern ; vierte am kiirzersten. Die Pinnule 
bleiben am ganzen Arm diinn und ihre Glieder, mit Ausnahme der 
2 basalen, stark verlingert. Linge einer Pinnula am circa 59. Arm- 
gliede 25 mm. 

Sacculi, spirlich. 

Fiirbung, hellbriunlich. 

Scheibe, etwa 12 mm. 

Klafterung, wenigstens 31 cm. (Exemplar St. 3357), 28-29 cm. Ex. 
St. 3424. 

Fundorte, St. 3357, in der Niihe von Mariato Point, 782 Faden 
Tiefe, Moderner Griinsand, Bodentemperatur 58,5 F.; und St. 3424, 
676 Faden, bei Las Tres Marias, griiner Sand, Globigerinen, Boden- 
temperatur 58,04 F. 

Die obige Beschreibung ist, mit Ausnahme der untersten Pinnule, 
welche zu verstiimmelt waren, und mit Ausnahme der Cirren, die 
leider ganz fehlten, auf das Exemplar von St. 3357 zu beziehen 
(Taf. 2, Fig. 15). Die Beschreibung der Cirren und Pinnule aber 
wurde nach den Exemplaren von Las Tres Marias entworfen. 

Ich habe die obige Schilderung der Art, die ich schrieb im 
Glauben, dass die vorliegenden Exemplare einer neuen Species ange- 
hérten, hier gegeben, weil sie von Carpenters Beschreibung und 
namentlich von seiner Abbildung, Chall. Rep. XXVI. Pl. 12, in 
vielen Puncten abweicht. Auf Carpenters Abbildung ist das Centro- 
dorsale ventral durch eine scharfe grade Linie begrenzt; von den 
ersten Radialien sind nur die Ecken sichtbar, die zweiten Radialia 
scheinen sich seitlich zu bertihren; sie sowohl wie die ersten Bra- 
chialia sind in Uebereinstimmung mit der Beschreibung lI. c. p. 148 
“deeply incised.” Alles das verhielt sich an meinen Exemplaren 
anders und veranlasste mich zuniichst zu einer specifischen Abtren- 
nung. Nachdem ich aber vor Kurzem vom Hamburger Museum ein 
von Dr. Rehberg in der Mageilhan Strasse (Smyth’s Channel) gesam- 
meltes Exemplar von Antedon rhomboidea selbst untersucht habe, bin 
ich ganz andrer Meinung geworden. Ich glaube, dass die vermeint- 
lichen Unterschiede nur auf Ungenauigkeiten der von Carpenter 
gegebenen Figur beruhen. Das Hamburger Exemplar von A. 
rhomboidea stimmt in jeder Hinsicht mit denen vom Albatross ge- 
sammelten. Die Art kommt also sowohl in der Magellhan Strasse 
als an der pacifischen Seite Central Americas vor. ‘A similar 
extensive geographical range from north to south has been observed 


140 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


in the distribution of some of the Mollusks Echini and Starfishes, 
which extend all the way from the southern extremity of South 
America to the Banamic region. The course of the northerly current 
setting along the west coast of South America must of course act as 
a distributer of the marine fauna of that region” (A. Agassiz, Bull. 
Mus. Comp. Zool., XXIII. No. 1, p. 75). 

Die Exemplare von Las Tres Marias unterscheiden sich nicht 
unbetrichtlich von dem Exemplar der St. 5357. Hierfiir ist viel- 
leicht maassgebend, dass sie aus sehr verschiedenen Tiefen stammen. — 
Die Las Tres Marias Exemplare haben vor Allem einen viel schlan- 
keren gracileren Habitus und erinnern in ihrer Erscheinung mit 
ihren dicht zusammengelegten, meist gestreckten Armen an eine 
Antedon phalangium, wie sie Carpenter (1. c. Taf. XX VIII) abgebildet 
hat. Die Knopfe der Armbasis und der Radialien sind nur schwach 
entwickelt und die alternirend seitlich gelegenen in der unteren 
Armgegend fehlen ganz. Die Armglheder, namentlich die syzygia- 
len, sind gestreckter. 

Die Abbildungen Taf. 1, Fig. 1-3, 6,10 und 11 geben ein Bild 
vom Calyx und seinen einzelnen Theilen. Fig. 1 zeigt das radiale 
Pentagon von der Dorsalseite. Weder von einem Basalstern noch 
von einer Rosette sind irgend welche Spuren zu bemerken. Die 
dorsale Oeffnung des Trichters ist weit (Fig, 1), und der Hohlraum 
im Centrodorsale (Fig. 2h) dementsprechend. Die Gelenkflichen 
der Radialia (Taf. 1, Fig. 3 und 10) zeigen im Gegensatz zu Antedon 
eschrichti (Chall. Rep. XXVI. Pl. 1, Fig. 8a) eine gleichmiissige 
Neigung gegen die verticale Hauptaxe; ihr ventraler Rand ist in der 
Mitte ziemlich stark eingeschnitten; von diesem Einschnitt ver- 
liiuft eine Verticalleiste gegen das Loch des Centraleanals zu. Die 
Muskelgruben sind grésser als die Ligamentgruben und von ihnen 
durch eine ziemlich horizontal gerichtete Querleiste geschieden, die 
mit 2 mehr oder minder deutlich ausgepriigten Wurzeln entspringt, 
deren eine von der Verticalleiste, die andre an der Seite des Cen- 
traleanals ihren Ursprung nimmt. Auf der Fliche der Muskel- 
gruven bemerkt man schwache, gebogene Querfurchen. Fig. 6 
zeigt die ventrale Oeffnung des Trichters und die kleinen in den- 
selben vorspringenden Zacken, die etwas unterhalb des Randes liegen 
und ventrale Enden von Liingsleisten sind, die sich auf der Trichter- 
flache der Radialia befinden (Taf. 1, Fig. 11). — Diese Verhiltnisse 
wurden an einem iilteren und einem viel jiingeren Exemplare von 
Las Tres Marias untersucht und tibereinstimmend gefunden. 


HARTLAUB: COMATULIDEN. 141 


Carpenter bildet im Chall. Rep. die besprochenen Theile fiir Ante- 
don eschrichti und antarctica ab. Von diesen zeigt Antedon eschrichti 
die meiste Uebereinstimmung mit unsrer Art. Wir finden auch bei 
ihm auf der ventralen Kante der Radialia den medianen Einschnitt, 
und scheint es, dass derselbe innerhalb der Gruppe allgemeiner vor- 
komnit, da er noch viel starker entwickelt ist an der neuen Species, 
die uns sogleich beschaftigen wird. Antedon eschrichti besitzt ferner 
Querfurchen in den Muskelgruben, aber was ihn wesentlich unter- 
scheidet, ist der Besitz einer Rosette, von der ich bei Antedon rhom- 
botdea nichts bemerkte. 

An vielen Exemplaren sassen cystenbildende Myzostomen. 


Antedon tanneri n. sp. 
Taf. I, Fig. 9; Taf, II. Fig. 13; Taf. III. Fig. 20, 22. 


Centrodorsale flach gewolbt, mit einer cirrusfreien Grube im 
Scheitel. Etwa 60 dichtstehende, gleichmiissig diinne, comprimirte 
Cirren, die eine Linge von 33 mm. erreichen. Die Cirren haben 
grossten Theils lingliche Glieder; die letzten davon sind kiirzer 
und haben manchmal einen endstiindigen kleinen Dorn auf der dor- 
salen Seite. 

Erste Radialia sichtbar, kurz. Zweite kurz und sich distalwiirts 
verschmalernd, nicht in Beriithrung untereinander. Axillare rhom- 
bisch, am Ansatz der Arme betrichtlich verbreitert. 

10 Arme, die sehr schlank und diinn sind und von Anfang an eine 
ganz unebene Oberfliche besitzen. Erste Brachialia von einander 
getrennt, kurz; zweite von unregelmissiger Form und von etwa 
doppelter Linge; drittes Glied (Syzygie) noch linger. Die fol- 
genden bis zum 7. sind annihernd quadratisch und haben stark ein- 
gebogene Flichen. Das 8. Glied hat die Liinge des dritten. Das erste 
und zweite Brachiale haben bisweilen einen kleinen scitlichen Dorn, 
der bei lezterem dicht vor dem Ansatz seiner Pinnula liegt. Auf das 
Glied der zweiten Syzygie folgen noch etwa 3 quadratische Glieder, 
dann wird die Form mehr trapezoid (aber nirgends dreieckig!). Die 
Glieder verbreitern sich alle etwas distalwirts, und ihre, in der 
zweiten Armhilfte etwas dornigen, Rinder iiberragen stark die 
Basis der nachfolgenden Gheder, was eine grosse Rauhigkeit der 
Armoberfliiche zur Folge hat. Die Arme sind an ihrer Basis rund- 
lich und weiterhin mehr comprimirt; sie verdiinnen sich bis etwa 
zum 15,—20. Gliede rasch und nachher ganz allmiilig. 


142 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


Erste Syzygie im 3. Brachiale, zweite im 8., dritte im 14., die fol- 
genden in Zwischenriiumen von 2-3 Gliedern, 

Pinnula des zweiten Brachiale 14-16 mm. lang mit 35-44 kurzen 
Gliedern, geisselformig und in eine sehr feine Spitze auslaufend. 
Am Ende der Pinnula ist eine Art Kamm entwickelt (Taf. 2, Fig. 
13). Pinnula des 4. Brachialia manchmal etwas linger sonst 
ebenso. Dritte Pinnula bedeutend kiirzer als die zweite und aus 
einer viel geringeren Zahl liinglicher Glieder zusammengesetzt ; 
ohne Kamm am Ende. Vierte Pinnula ebenso lang, dann nimmt 
die Linge ganz allmiilig ab. Die 14 Pinnula ist etwa 10 mm. lang, 
doch sind die der aéusseren Armhialfte wieder einige mm. langer. 
Alle diese Pinnule sind sehr diinn und haben, mit Ausnahme der 
zwei basalen, stark verlingerte Glieder. 

Scheibe etwa 7 mm. Dm., mit zerstreuten Kalkkérperchen. — Mund 
central. 

Sacculi zahlreich an den Ambulacren der Pinnule, Arme und 
Scheibe. 

Klafterung durchschnittlich 20 mm. 

Férbung, in Alcohol, hell gelblich; Pinnulz briunlich., 

Fundort, St. 3385, Eingang der Bucht von Panama, 286 Faden, 
grimer Mud; Bodentemperatur 45,9 F. Zahlreiche Exemplare. 


In einiger Hinsicht gleicht diese, neue Art vollkommen der vori- 
gen, so besonders in der Form ihres Centrodorsale und ihrer Cirren, 
doch lisst ihr ganz anderer Habitus, die grosse Rauhigkeit ihrer 
Armoberfliichen und die Verschiedenheit der radialen Gelenkfliche, 
die ich an 2 Exemplaren untersuchte, wohl keinen Zweifel an ihrer 
Berechtigung zu. Beziiglich der Gelenkflichen vergleiche man die 
Figuren 9 und 10 auf Tafel 1. Wir finden bei unsrer Art an Stelle 
einer verticalen Lingsleiste, eine breite flache intermuskulare 
Mulde. Solch eine Bildung ist charakteristisch fir die Gattung 
Actinometra, bei Antedon aber sehr selten (Antedon disciformis Pal- 
mata Gruppe). Sie gewinnt fiir unsre Art an Interesse, wenn man 
bedenkt, dass auch die unteren Pinnule durch den Besitz eines 
Kammes ein Actinometra iihnliches Verhalten zeigen. Von den 
radialen Gelenkflichen wiire weiterhin der ungemein tiefe Ein- 
schnitt ihres ventralen Randes zu erwaihnen. Zu beiden Seiten 
dieses Einschnittes liegen die Muskelgruben auf leicht nach aussen 
gekriimmten Fortsiitzen, die in ihrer Form langen Ohren gleichen. 
Sie haben leicht angedeutete, gebogene Querleisten. Von den Liga- 


HARTLAUB: COMATULIDEN. 143 


mentgruben sind sie durch sehr schwache Leisten getrennt, die 
ihren Ursprung seitlich vom Centraleanal nehmen, anfiinglich ver- 
tical verlaufen und dann in einem ziemlich scharfen Winkel schriig 
nach oben abbiegen. 

Das starke Ueberragen der Armglieder (overlapping of the arm- 
joints), was unsrer neuen Art ein so eigenes Gepriige verleiht, 
theilt sie in der Eschrichti Gruppe mit Antedon antarctica Carp. 

An Zierlichkeit des Wachsthums wird sie wohl von Keiner der 
verwandten Formen erreicht. 

An sehr vielen Exemplaren fand ich cystenbildende Myzostomen. 


TENELLA GRUPPE Carp. Cuatu. Rep. XXVI. p. 156. 


“10 Arme. Die Glieder der untersten Pinnule, welche oft lang 
und schlank sind, sind linger wie breit und dies oft in hohem 
Grade.” 


Die von Carpenter in der Tenella Gruppe zusammengefassten 
Arten, einige zwanzig an der Zahl, haben ausserordentlich weite 
Grenzen sowohl in ihrer horizontalen als in ihrer bathymetrischen 
Verbreitung. Es sind unter ihnen zwei (Antedon hirsuta und exigua) 
vom 46. Grade siidlicher Breite und eine (Antedon prolixa) vom etwa 
70. Grade nordlicher Breite. Nur sehr wenige Antedon Arten iiber- 
schreiten tiberhaupt diese Breiten. Andrerseits ist die Hauptmenge 
der bekannten Species atlantisch, finf aber gehdren dem westl. 
Pacific an und eine Art (Antedon nana) dem indischen Archipel 
(Amboina). Die Arten, welche ich hier zu beschreiben habe, sind 
die ersten aus dem ostlichen Theile des Stillen Oceans. Bathyme- 
trisch gehéren zur Tenella Gruppe sowohl littorale Species wie z. B. 
Antedon nana Hartl. und die europiiische Antedon rosacea, als auch 
Antedon abyssicola aus 2900 Faden, der gréssten Tiefe, aus der iiber- 
haupt Comatuliden gefischt wurden. Die neue Art Antedon parvula 
zeigt viel Aehnlichkeit mit der ebenfalls pacifischen Antedon alter- 
nata (south of Japan, northeast of New Zealand, Kermadecs, north 
of Papua) wihrend Antedon bigradata nu. sp. niher mit der oben er- 
wihnten Antedon prolixa von Faeroe Channel und Smith Sound, 
Kara See, verwandt ist. Wie die iibrigen pacifischen Arten, so 
wurden auch die vom Albatross gefischten in grossen Tiefen gefan- 
gen nimlich Antedon parvula in 978 Faden und Antedon bigradata in 
385 und in 555 Faden. Ausser diesen beiden Species erhielt der 


VOL. XXVII. — NO. 4. 2 


144 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


Albatross noch stark verstiimmelte Exemplare von zwei andern 
Arten, von denen das eine, wahrscheinlich Antedon abyssicola, bei St. 
39381 aus der bedeutenden Tiefe von 1772 Faden gefischt wurde. 


Antedon parvula n. sp. 
Taf. III. Fig. 21. 


Centrodorsale annihernd conisch mit schwacher Wolbung nach 
aussen. 20-30 Cirren, von 5-6 mm. Linge, deren Gliederzahl etwa 
20 betraigt. Das 3., 4. und 5. Glied sind stark verlingert. Die fol- 
genden werden schnell kiirzer; die langen Glieder sind in der Mitte 
eingeschniirt. Die distalen Rinder simmtlicher Glieder springen 
vor, und die dorsale Contur des Cirrus ist stark gezackt. Das 
Ende des Cirrus ist etwas comprimirt. 

Erstes Radiale etwas kiivzer als das zweite und mit leicht eingebo- 
genem distalem Rande; seitlich frei. Zweite kurz, nicht einge- 
schnitten, ebenfalls seitlich frei. Axillare rhombisch. 

10 Arme. Erste Brachialia kurz, scheibenférmig, untereinander 
nicht in Bertihrung. Zweite fast doppelt so lang, von etwas wech- 
selnder Form (bald mehr dreieckig, bald mehr viereckig). Drittes 
Brachiale (Syzygie) noch linger; jedes seiner syzygialen Glieder 
reichlich so lang wie das erste Brachiale. Es folgen vier fast quadra- 
tische Glieder und darauf das 8. (Syzygie), das wieder linger ist; 
dann ausschliesslich trapezoide Glieder, die bald die definitive, nicht 
unbedeutende Linge erreichen. (Das Armende ist an keinem Arm 
erhalten.) 

Syzygien im 3., 8., 12. und darauf in Unterbrechungen von einem 
Gliede. 

Pinnula des 2. Brachiale sehr diinn ; die Glieder sind in der Mitte 
und gegen das Ende der Pinnula zu ziemlich stark verlingert, und 
haben vorspringende, fein gezihnte distale Riinder. (Keine war 
vollstiindig, eine aber bis zu 15 Gliedern erhalten). Die folgende 
Pinnula (Br. 6) 6 mm. lang mit circa 15 Gliedern, die der Mehrzahl 
noch stark verlingert sind. Pinnula des 6. Brachiale kirzer und 
aus etwa 10 stark verlingerten Gliedern bestehend. Die Pinnula 
des 8. Brachiale scheint die kiirzeste zu sein. Die Glieder simmt- 
licher folgenden Pinnulz sind stark verlingert. 

Schetbe nicht sichtbar (auf etwa 3 mm. Dm. zu schiitzen). 

Farbung weisslich. 

Klafterung auf 60 mm, zu schiitzen. 


HARTLAUB: COMATULIDEN. 145 


Sacculi nicht deutlich zu erkennen. 

Fundort, St. 8363, 978 Faden, Cocos Islands, weisser Globigerinen 
Schlamm ; Bodentemperatur 37,5 F. Ein Exemplar. 

Das winzige Exemplar, welches obiger Beschreibung zu Grunde 
liegt, trenne ich trotz iibrigens grosser Aehnlichkeit mit Antedon 
alternata Carp. vorliufig von dieser specifisch ab, weil sich seine 
Cirren sehr wesentlich von denen jener unterscheiden (Taf. III, Fig. 
21, und Chall. Rep. Pl. 22, Fig.8 & 9). Ich kann aber nicht liugnen, 
dass mir trotzdem diese neue Art einiges Bedenken macht. Gewisse 
Uebereinstimmungen, so vor Allem solche in der Form des Centro- 
dorsale, in der Form der Radialia, in der abwechselnden, ein Glied 
tiberspringenden Lage der Syzygieen, sind auffallend genug. 


Antedon bigradata n. sp. 
Taf. I. Fig. 5. 


Centrodorsale ziemlich lang conisch, mit abgestumpfter Spitze und 
etwas gewolbten Seiten. Die Cirrusgruben sind tief (Cirren selbst 
sind nicht erhalten), etwa 80 an der Zahl und stehen in 5 Feldern, 
die durch schmale interradiale Zwischenstreifen getrennt sind. 
Innerhalb der Felder stehen die Cirrusgruben in 4 verticalen Reihen, 
jede Reihe mit 4 Gruben. Die einzelnen Gruben berithren sich nicht 
untereinander. 

Zwischen Centrodorsale und jedem ersten Radiale befindet sich 
eine spaltartige Vertiefung. Erste Radialia sichtbar und zwar nicht 
bloss an den Ecken des Calyx (wie die A. hystrix). Zweite Radiale 
kurz; es steht senkrecht zur Hauptaxe und im rechten Winkel zum 
Axillare; ebenso steht das erste Brachiale zum zweiten Brachiale. 
Kelch und Armbasis erscheinen durch diese Stellungsweise auffal- 
lend getreppt. Das radiale Axillare ist viereckig, sein proximaler 
Winkel spitz und lang ausgezogen; die beiden Seitenwinkel nach 
vorn geriickt. 

Erste Brachiale kurz und dorsal kiirzer als ventral. Zweite Bra- 
chiale dreieckig, drittes ziemlich kurz, dann 4 ebenfalls ziemlich 
kurze trapezoide Glider, die auf ihrer pinnulatragenden Seite etwa 
halb so kurz sind als auf der andern. 

Die Pinnula des zweiten Brachiale hat etwa 13 Glieder, von denen 
schon das erste bedeutend linger als dick ist, das zweite aber schon 
mindestens 2 mm. lang ist. Die Pinnula des 3. Brachiale ist klein 
und hat ebenfalls ein langliches Basalglied. Pinnula des 4. Brachiale 


146 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


kiirzer wie die des 2. mit 9 Gliedern, von denen das Basale ebenfalls 
linglich ist. An der Pinnula des 6. Brachiale, die aus etwa 9 Glie- 
dern besteht, sind die zwei basalen Glieder kaum noch linger wie 
breit und auch die folgenden Glieden etwas kiirzer. 

Scheibe des Exemplars von St. 3404 hat 12 mm. Dm. 

Férbung, hell réthlich braun mit weissen Flecken auf den Ver- 
bindungen der Pinnula Glieder. 

Fundorte, St. 8358, 555 Faden, in der Nihe von Cap Mariato 
Point, “ Modern Greensand,” Bodentemperatur 40,2 F.; St. 3404, 
885 Faden, zwischen Chatham und Hood Island, felsiger Grund, 
Bodentemperatur 43,2 F. 

Die neue Art ist nahe verwandt mit Antedon prolixa Sladen, und 
mit dieser ist, wie mir Carpenter brieflich mittheilte, Antedon hystrix 
identisch. Da mir die Sladensche Beschreibung von A, prolixa nicht 
zugiinglich ist, beschrinke ich mich darauf auf die Unterschiede hin- 
zuweisen, welche die neue Art im Vergleich mit Carpenters Beschrei- 
bung von A. hystrix darbietet. Besonders ist da hervorzuheben, dass 
die Cirren von A. bigradata in 5 Feldern stehen und innerhalb dieser 
wieder in Verticalreihen; sodann ist die spaltartige Vertiefung 
zwischen jedem ersten Radiale und dem Centrodorsale bemerkens- 
werth. Mehr Gewicht aber ist wohl noch auf die Verschiedenheit 
zu legen, welche die Pinnula des 2. Brachiale im Vergleich mit der 
von A. hystrix zeigt. Wihrend sie niimlich bei letzterer 15 mm. 
lang ist und einige 30 Gheder hat “the first six of which are short 
and nearly square,” hat sie bei unserer neuen Art 13 Glieder, von 
denen schon das erste bedeutend linger als dick ist. 


Antedon spec. 
Taf. IV. Fig. 25. 


Fundort, St. 3381, 1772 fath., Golf von Panama, in der Nahe von 
Malpelo Island, “Green Mud,” Bodentemperatur 37,2 F. Ein 
Exemplar. 

Am vorliegenden Exemplar fehlen die Cirren, und die Arme sind 
vom 3. Brachiale ab abgebrochen. Von den untersten Pinnule 
sind nur stellenweise einige Glieder erhalten. 

ts handelt sich wahrscheinlich um Antedon abyssicola oder eine 
dieser sehr nahe verwandten Art. 

Das Centrodorsale ist conisch und trigt etwa 25 Cirrusgruben, die 
relativ gross sind und dicht aneinander stehen. (Antedon abyssicola 


HARTLAUB: COMATULIDEN. 147 


hat nach Carpenter 15 Cirren). Die ersten Radialia sind wie bei 
A. abyssicola vollkommen sichtbar. Die Ecken schieben sich ein 
wenig zwischen die zweiten Radialia ein, sodass diese nicht seitlich 
in Beriihrung stehen. Dasselbe Verhalten zeigt A. abyssicola gele- 
gentlich in ausgeprigtem Maasse. 

An dem Exemplar sitzt als Schmarotzer festgeheftet ein Stylifer 
verwandte Schneckenart (nach Prof. v. Martens wahrscheinlich eine 
Mucronalia). 

Artedon spec. 


Taf. IV. Fig. 27. 


Fundort, St. 8354, 322 fath., in der Nihe der Kiiste bei Mariato 
Point, “Green Mud,” Bodentemperatur 46,0 F. Ein Exemplar. 

Eine Bestimmung ist nicht ausfiihrbar, weil die Cirren und unter- 
sten Pinnule fehlen und die Arme simmtlich nahe ihrer Basis abge- 
brochen sind. Die conische Form des Centrodorsale, die bedeutende 
Zahl der Cirrusgruben sowie die Form der Radialia und untersten 
Armglieder lassen auf eine Antedon prolixa nahestehende Art wenn 
nicht gar auf diese selbst schliessen. 


Uebersicht der vom Albatross gesammelten Arten. 


Gesammtzahl der Species, 7. 
Siimmtliche Species gehéren dem Genus Antedon an und verthei- 
len sich, wie folgt, auf drei Gruppen : — 


I. BASICURVA GRUPPE (arp. 


Antedon agassizii n. sp. 


Fundorte : 
St. 3357, 782 Faden, “modern greensand,” nicht weit von Mariato Point. 
« 3408, 684 “  Globig. Ooze. Galapagos 
“ 3409, 327 “ “black sand.” rey 


II. ESCHRICHTI GRUPPE Carp. 


Antedon rhomboidea Carp. 
Fundorte : 


St. 3357, 782 Faden, “‘ modern greensand,” nicht weit von Mariato Point. 
* 3424, 676 “ “gray sand,” Las Tres Marias Inseln. 


Antedon tanneri n. sp. 
Fundort: 


St. 3385, 286 Faden, “green mud,” Golf von Panama. 


148 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


III. TENELLA GRUPPE Carp. 


Antedon spec. 
Fundort: 
St. 3354, 322 Faden, “green mud,” bei Mariato Point. 


Antedon bigradata n. sp. 
Fundorte : 
St. 3358, 555 Faden, “ modern greensand,” nicht weit von Mariato Point. 
« 3404, 385 “ ‘rocks,’ Galapagos. 


Antedon parvula n. sp. 
Fundort : 
St. 3363, 978 Faden, ‘ white Glob. Ooze.,” Cocos Inseln. 


Antedon spec. (abyssicola Carp. ?). 
Fundort : 
St. 3381, 1772 Faden, “ green mud,” nicht weit von Malpelo Island. 


Besonderes Interesse verdienen des Fundortes wegen die zur 
Eschrichtt Gruppe gehorenden Arten. 


HARTLAUB: COMATULIDEN. 149 


ERKLARUNG DER ABBILDUNGEN. 


TAFEL I. 


Fig. 1. Antedon rhomboidea Carp. 


Fig. D) “ “ 

Fig. 3 “ “ec 

Fig. 4 ” agassizii n. sp. 
Fig. 5 ‘ bigradata n. sp. 
Fig. 6 ae rhomboidea n. sp. 
Hips 7 ee agassizii n. sp. 
Fig. g “ “ 

Fig. 9 “ tanneri n. sp. 

Fig. 10 as rhomboidea 

Fig. 1 1 “ “ 


Exemplar von Las Tres Marias. Dor- 
sale Ansicht des radialen Pentagons ; 
vergrossert X 5. 

Ventrale Ansicht des Centrodorsale vom 
selben Exemplar; vergr. X 5. 

Calyx von der Seite, vom selben Exem- 
plar; vergr. X 5. 

Ventrale Ansicht des Centrodorsale ; vergr. 

X 7. 

vergr. X 3. 

Ventrale Ansicht des Calyx ; vergr. x 5. 

Dorsale Ansicht des radialen Pentagons 
vom selben Exemplar wie Fig. 4; 
vergr. X 7. r. Rosette; 6. ein Strahl 
des Basalsterns. 

Seitenansicht des Calyx; vergr. X 6. m. 
Muskelgrube ; /. Ligamentgrube. 
Gelenkflache des ersten Radiale; vergr. 

x 15. 

Gelenkflache des ersten Radiale; vergr. 
om Is 

Ansicht eines Radiale vom Trichter aus; 
vergr. X 6. 


150 


Fig. 
Fig. 


Fig. 
Fig. 


Fig. 
Fig. 


Fig. 
Fig. 


Fig. 
Fig. 
Fig. 
Fig. 


5 


Fig. 


Fig. 25. Antedon spec. abyssicola 
Carp.? mit einer daran sitzenden 
Mucronalia ? 


12. Antedon rhomboidea n. sp. 
13. 


14. 
15. 


16. 
fe 


BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


“ce 


TAFEL II. 


tanneri Nn. sp. 


rhomboidea n. sp. 


“ 


agassizli n. sp. 


rhomboidea n. sp. 


agassizii N. sp. 


“ 


Pinnula des zweiten Brachiale ; vergr. X 4. 

Ende der Pinnula des zweiten Brachiale ; 
stark vergr. 

Theil eines jungen Exemplars von Las 
Tres Marias; vergr. X 4. 

Theil eines Exemplars von St. 3357 ; vergr. 
X 23. 

Stiick aus der ausseren Armhilfte; vergr. 

Cirrus eines Exemplars von Las Tres 
Marias; vergr. X 2. 

Proximale Armregion; vergr. 

Glied einer Pinnula von der Seite; sehr 
stark vergr. se, se’ Seitenplatten; s. 
Platz des Sacculus; ¢. Liicke fiir den 
Tentakeldurchtritt ; d. Deckplatten. 


TAFEL II. 


. Antedon tanneri n. sp. 


“ 


“ 


parvula n. sp. 
tanneri 0. sp. 


agassizii n. sp. 
rhomboidea. 


vergr. X 1}. 

Cirrus ; vergr. X 11. 

Stiick aus der mittleren Armgegend ; 
vergr. X 4. 

Glied einer Pinnula; stark vergr. 

Exemplar von Las Tres Marias; vergr. 
x12. 


TAFEL IV. 


Fig. 26. Antedon agassizii n. sp. 
Fig. 27. ye 


spec. (Tenella Gruppe). 


vergr. X 41. 
vergr. X 2. Peters del. 


vergr. 43. 


HARTLAUB: COMATULIDEN. 151 


Im Anhang an die Beschreibung der Albatross Crinoiden méchte 
ich noch einen ebenfalls dem Mus. of Comp. Zoédlogy gehérenden 
Antedon von Gaspard Sir. beschreiben, den ich fiir neu erachte. 


Antedon subtilis n. spec. (Palmata Gruppe). 


Centrodorsale flach scheibenformig, mit etwa 20 glatten Cirren am 
Rande, die meist zweireihig stehen. Cirren etwa 12 mm. lang, ziem- 
lich diinn und in der ausseren Hilfte comprimirt. Circa 20 Cirrus- 
glieder, die vom 5. an etwas lianglich sind; die dusseren Glider 
haben einen ganz schwachen Dorn, das vorletzte einen stirkeren. 

Radien vom ersten Radiale an ganz frei seitlich. Erste Radialia 
seitlich etwas sichtbar. Zweite Radialia kurz, etwas kiirzer wie 
die Seiten des fiinfeckigen Radiale axillare. Distichale und pal- 
mare Stiimme zweigliedrig. Erste Distichale und Palmare etwa so 
lang wie die Seiten des 2. u. 3. Radiale zusammengenommen. Keine 
Postpalmaria. Die Glieder der Radialia, Stamme und Arme voll- 
kommen glatt. Nirgends Buckel auf den Verbindungen der Axil- 
laria oder sonstige Protuberanzen. 

20 Arme. (Ein Radius mit fiinf Armen, einer mit drei, die iibri- 
gen mit vier). Die Arme sind ziemlich diinn, vollkommen glatt, 
etwa 5cm. lang und mit sehr feinen Pinnule besetzt; die Arm- 
glieder sind kurz und zwar bis zum 8. mehr oder minder scheiben- 
formig, vom 9. an aber ausgesprochen dreieckig; so bleiben sie bis 
etwa zum 20, Gliede, von da ab wird die Form mehr trapezoid, und 
spiter quadratisch. Das erste Brachiale steht in theilweisem Con- 
tact mit dem Nachbargliede. 

Erste Syzygie im 3. Brachiale, zweite im 14. Brachiale, die dritte 
nach einen Zwischenraum von 5-6 Gliedern, die folgenden in 
Zwischenriiumen von 6-7. 

Pinnula des 2. Brachiale etwa 7 mm. lang mit circa 13 glatten 
Gliedern, die mit Ausnahme der 2 basalen linglich sind. Pinnula des 
4. Brachiale betrachtlich stiirker und 10 mm. lang, geisselférmig. 
Glieder wie bei der vorigen liinglich mit Ausnahme der zwei ersten. 
Die darauf folgenden 3 Pinnule derselben Armseite ganz winzig, 
etwa 2mm. lang. Dann nimmt die Linge allmalig etwas zu, doch 


aay BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


bleiben die Pinnulz des Mittelarms klein und diinn. Die Pinnule 
des Armendes sind von haarartiger Feinheit, aber etwas linger 
(5 mm.), die Glieder stets langlich. 

Sacculi dichtstehend. 

Klafterung etwa 103 cm. 

Scheibe tief eingeschnitten. 

Firbung gleichmissig weiss. 

Fundort, Gaspar Str. Ein Exemplar. 


Die Art ist nahe verwandt der von mir beschriebenen Art Ant. 
Klunzingeri von Koseir aus dem Rothen Meer, unterscheidet sich 
aber von dieser durch die Kleinheit der Pinnule des 6. Brachiale. 
Dieselbe ist bei A. Klunzingeri so lang wie die des ersten Brachiale. 
Moglicher Weise wird sich nach Untersuchung grésseren Materials 
herausstellen, dass die beiden Arten identisch sind. 


CRINOIDEA PL.I. 


Lith. Werner & Winter, Frankfort’”M 


10. 


ALBATROSS ExXP. 1891. 


Q.Peters del. 


CRINOIDEA PLLII. 


ALBATROSS ExXpP. 1891 


S — ae ge = 
™~ 


} } Se by} 
SON Vege, 
} Sf) ‘4 ea = 
ae Ke 3 
vy 
i 4 eae a. a i a a 
az =—=__—-@___ = 


works er roa 


Werner & Winter, Frankfort °/M. 


Lith 


35) 
Ss. cel. 


0. Peter 


CRINOIDEA PLLIII. 
4 


a= Ae ee es Oe see 


On ee em aati eal a ee 


ALBATROSS EXP. 1891. 


Lith.Werner& Winter, Frankfort 2M 


O.Peters del. 


4 ALBATROSS ExP. 1891. CRINOIDEA PLLIV. 


f 


—— 


Werner&Winter, Frankfort ”M 


Lith 


Hartlaub u.0.Peters del. 


3 
_ 


RTS ON THE DREDGING OPERATIONS OFF THE WEST COAST OF 
ENTRAL AMERICA TO THE GALAPAGOS, TO THE WEST COAST 
OF MEXICO, AND IN THE GULF OF CALIFORNIA, IN CHARGE OF 
ALEXANDER AGASSIZ, CARRIED ON BY THE U.S. FISH COMMIS- 
SION STEAMER “ALBATROSS,” DURING 1891, LIEUT. COMMANDER 
4. L. TANNER, U. S. N., COMMANDING. 


XIX. 
DIE OSTRACODEN. 


Von G. W. MuLLeER: 


a {Published by Permission of MARSHALL McDonaLp, U.S. Fish Commissioner.] 


s 


With THREE PLATEs. 


CAMBRIDGE, MASS., U.S. A.: 


PRINTED FOR THE MUSEUM. 
OctToser, 1895. 


AT HARVARD COLLEGE. Ny ( 


We) 
We 
Vou. XXVII. No. 5. | Aig 
ly 
AG 


Bulletin of the Museum of Comparative Zoology 
AT HARVARD COLLEGE. 
Vout. XXVIII. No. 5. 


REPORTS ON THE DREDGING OPERATIONS OFF THE WEST COAST OF 
CENTRAL AMERICA TO THE GALAPAGOS, TO THE WEST COAST 
OF MEXICO, AND IN THE GULF OF CALIFORNIA, IN CHARGE OF 
ALEXANDER AGASSIZ, CARRIED ON BY THE U.S. FISH COMMIS- 
SION STEAMER “ALBATROSS,” DURING 1891, LIEUT. COMMANDER 
Z. L. TANNER, U.S. N., COMMANDING. 


XIX. 
DIE OSTRACODEN. 


Von G. W. Mutter. 


(Published by Permission of MARSHALL McDONALD, U. S. Fish Commissioner.] 


“Wirnh THREE PULAtTEs. 


CAMBRIDGE, MASS., U.S. A.: 


PRINTED FOR THE MUSEUM. 
OcropER, 1895. 


4 


OCT18 1895 


No. 5. — Reports on the Dredging Operations off the West Coast 
of Central America to the Galapagos, to the West Coast of 
Mexico, and in the Gulf of California, in charge of ALEXANDER 
AaassiZ, carried on by the U. S. Fish Commission Steamer 
«“ Albatross,” during 1891, Lieut.-Commander Z. L. TANNER, 
U.S. N., Commanding. 


[Published by Permission of Marsuatit McDona p, U. 8S. Fish Commissioner.] 
XIX. 
Die Ostracoden. Von G. W. MUuuer. 


Die Zahl der gesammelten Arten ist nur gering: zwei Cypridiniden 
und vier Halocypriden, doch kniipft sich an die beiden Cypridiniden als 
Vertreter einer neuen Gattung von sehr abweichendem K6rperbau und 
ebenso abweichender Lebensweise ein besonderes Interesse. Es handelt 
sich, was ich zum Verstiindniss der folgenden Beschreibung vorausschicke, 
um Arten von ausschliesslich pelagischer Lebensweise. Ich lege der 
Besprechung der Cypridiniden, fiir die ich mit Ricksicht auf ihre auf- 
fallende Grosse den Gattungsnamen Gigantocypris wiihle, die haufigere 
der beiden Arten, Gigantocypris Agassizi?, zu Grunde. 

Bei einer oberflachlichen Betrachtung kann man in Zweifel sein, ob man 
es mit einem Ostracoden zu thun hat. Zuniichst tiberrascht die ausser- 
ordentliche Grésse, wie sie sonst von lebenden Ostracoden nicht entfernt 
erreicht wird, wie sie sich auch nur ganz ausnahmsweise bei fossilen 
findet. Die Thiere erreichen eine Linge von 23 mm. bei eine Hohe 
von 19.5 und eine Breite von 18 mm. Weiter bietet das Thier das 
Aussehen einer grossen, diinnwandigen, durchscheinenden Blase mit 
_ kurzer, schlitzformiger Oeffnung, nicht den eines Thieres mit zweiklap- 
piger Schale (Tafel 1, Fig. 1, 2). Eigentlich kann man auch nicht 
mehr von einer zweiklappigen Schale sprechen. Wiéahrend sonst bei den 
_ Ostracoden die Strecke, fiir welche beide Schalenhalften mit einander 
~ verbunden sind, etwa 4, stets aber weniger als } der Peripherie betragt, 
betriigt sie hier etwa 2, nur } der Schalenrander ist frei. Die freien Rander 


der Schale legen sich dicht~ aneinander, nur am vorderen und hinteren 
VOL. XXVII. — NO. 5. 1 


156 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


Ende des Schlitzes weichen sie deutlich auseinander, vorn zur Bildung der 
bekannten Rostralincisur (Tafel 1, Fig. 1, 2, 6, 8), hinten ftir eine ovale 
Oeffnung, welche den anderen Cypridiniden fehlt (Tafel 1, Fig. 1, 2, 7, 9). 
Innerhalb der tibrigen 2 der Peripherie, wo beide Schalenhialften fest mit 
einander verbunden sind, zeigt sich nur in der Nachbarschaft der 
genannten Oeffnungen eine deutliche Abgrenzung zweier Schalenhilften, 
sonst markirt sich die Grenze nur als feine, schwer aufzufindende 
Linie. 

Uebrigens ist die Schale sehr diinnhautig, nur die freien Rander sind 
verstiirkt. Die Schale ist so diinn und azart, dass fast simmtliche 
Thiere, die ich erhielt, sehr stark, auf 4 oder weniger ihres Volums 
zusammengedriickt waren, wofiir freilich nicht nur die geringe Wider- 
standsfiihigkeit der Schale, sondern auch die geringe Menge fester 
Substanz, das ausserordentliche Ueberwiegen der Blutriume verantwort- 
lich zu machen ist. Vermuthlich ist dieses Zusammendriicken bereits 
im Netze erfolgt, die Thiere haben dem Druck des Wassers nicht wi- 
dersteben kénnen. Nur 1 Individuum war in seiner Korperform gut 
erhalten, 1 zweites leidlich, alle ibrigen waren stark zusammengedriickt. 

Von Verkalkung ist an der Schale durchaus nichts zu bemerken ; 
auch von krystallinischen Concretionen, wie man sie in Folge der Auf- 
lésung und Wiederausscheidung des Kalkes in der Schale oder an anderen 
Korpertheilen bei conservirten Halocypriden und Cypridiniden findet, 
habe ich nichts entdecken kénnen. Ich glaube danach, dass jede Kalk- 
ablagerung fehlt, ein Schluss, der durch das, was ich eben tiber das 
Zusammendriicken der Schale sagte, bestatigt wird. 

Die Schale ist glatt, entbehrt jeder Skulptur; Haare fehlen so gut 
wie ganz, nur in der Rostralincisur und in ihrer Nachbarschaft findet 
sich am Rand ein dichte Reihe kurzer, starker Borsten (Tafel 1, Fig. 8). 
Das einzige wohl erhaltene Individuum zeigte einen grtinlichen Schim- 
mer. 

Die innere Schalenlamelle ist noch viel zarter als die 4ussere, entfernt 
sich weit von ihr (vergl. Tafel 1, Fig 17. Z., a. Z.), so dass der Schalenraum 


einen sehr umfangreichen Blutraum darstellt. Beide Schalenlamellen 


sind durch sehr zahlreiche feine Bindegewebsfasern mit einander ver- 
bunden, welche die Schale fein und dicht radiar (nach dem Centrum des 
Korpers hin) gestreift erscheinen lassen (siehe Fig. 1). Der Verlauf des 
Innenrandes (Jr.) in der Nachbarschaft der Rostralincisur ist aus Tafel 1, 
Fig. 8 ersichtlich. Am Ventralrand zieht er dem Schalenrand parallel, 
in geringer Entfernung von demselben. Zu einer deutlichen Verschmel- 
zung beider Lamellen kommt es nicht. Der Saum ist schmal, ganzran- 


— 


MULLER: OSTRACODEN. 157 


dig oder fein gesiigt, gestreift. In der hinteren Hialfte des Schlitzes 

zieht eine verstiirkte Rippe dem eigentlichen Schalenrand parallel, nach 

innen von demselben und in seiner niichsten Nahe (Tafel 1, Fig. 7, 

9 F,). Kleine einzellige Driisen sind tiber die ganze Schale zerstreut ; 

etwas reichlicher entwickelt sind sie am Schalenrand ; die Nerven der 

Schale sind viel schwiicher entwickelt als bei den anderen Cypridiniden. 

Bei der Untersuchung des eigentlichen Koérpers thun wir gut, uns 
iiber die Lage der Organe zu orientiren, ohne das Thier aus der Schale 
herauszulésen, und das geht einigermaassen, da die Schale ziemlich 

_ durchsichtig ist. Wir vermégen sehr wohl Herz (C.), Frontalorgan 

(Fr.), Schliessmuskel (J7.), Darm (D.), Furcalfeld (Hf), 2. Antenne 

(An. 2), Putzfuss (Pf.), sowie den Riicken des Thieres mit seinen Mus- 

keln zu erkennen (Tafel 1, Fig. 1, 2). Die nicht genannten Gliedmaas- 
sen liegen als unentwirrbare Masse auf den kleinen Raum zwischen 
2 Antenne und vorderer Grenze des Furcalfeldes zusammengedringt. Wie 
aus der Figur 1 ersichtlich, ttberwiegt die Riickenpartie des K6rpers 
_ sehr stark, diese gréssere hintere Halfte wird nur zum kleinsten Theil 

erfillt von dem Magen und den Geschlechtsorganen, der Rest stellt 
einen ausserordentlich umfangreichen Blutraum dar. Bei dem ge- 
zeichneten Thier, wie bei allen untersuchten der Art, war der Magen 
: leer und stark contrahirt (vergl. unten), bei gefiilltem Magen wird sich 
_ das Verhiltniss etwas zu Ungunsten des Blutraumes verschieben, 
: immerhin zeigt sich das, was schon bei Besprechung der Schale erwéihnt 
_ wurde, doch noch viel auffiilliger als dort: Die Blutraéume haben einen 
_ ausserordentlichen Umfang erlangt, ihnen gegeniiber tritt die feste Sub- 
i stanz des Kérpers sehr zuriick. Es ist mir aus der Gruppe der Krebse 
: kein Beispiel fiir ein abnliches Ueberwiegen der Blutréume bekannt 
_ geworden. Versuchen wir das Thier aus der Schale auszulésen, so 
} erfolgt beim Anschneiden der Schale ein starker Ausfluss der Korper- 
_ fliissigkeit, beide Schalenlamellen legen sich dicht aneinander, der 
Korper fallt zusammen; augenscheinlich geniigt beim Fangen eine 
geringe Verletzung des Thieres, um das Blut ausfliessen zu lassen und 
den KG6rper arg zu entstellen. 

Gliedmaassen. — Allgemein lasst sich von der Mehrzahl der Glied- 
maassen sagen, dass sie verhiltnissmissig kiirzer sind als bei anderen 
Cypridiniden (vergl. Tafel 3). Die 1. Antenne (Tafel 3, Fig. 2) ist 
gestreckt und ziemlich schlank, sie ist 7 gliedrig, das 5. Glied tragt die 
gefiederte Sinnesborste der Cypridiniden ; dieselbe ist in beiden Ge- 
schlechtern gleich stark entwickelt, die Fiederung ist nicht tbrig reich. 
Das letzte Glied, an dem sich Reste einer Verschmelzung aus 7 und § 


158 BULLETIN : MUSEUM OF COMPARATIVE ZGOLOGY. 


nicht nachweisen lassen, tragt drei lange, spiarlich gefiederte, starke 
Tastborsten, die beim 9 etwa 4, beim g tiber 2 mal so lang als die 
Glieder 2-7, 2 kiirzere Sinnesborsten, 1 kirzere starke und eine sehr 
kurze schwache Borste. Beim ¢ (Tafel 1, Fig. 24) tragen einzelne 
Borsten ahnliche Zweige mit saugnapfartigen Gebilden wie bei Cypri- 
dina mediterranea ; jeder Zweig tragt nur einen kleinen Napf nahe der 
Basis, ist ibrigens einfach. Solcher Zweige tragt die eine starke Tast- 
borste 2, die kiirzere 5. Die 2. Antenne (Tafel 3, Fig. 1) erinnert im | 
Bau, wie die 1., lebhaft an die Gattung Cypridina, die Borste des 2. 
Gliedes des Schwimm oder Aussenastes ist kurz, ungefiedert, unbedornt, 
die ibrigen Borsten sind sehr lang, gefiedert, nicht bedornt ; der Dorn, 
der bei Cypridina neben der Basis der Borsten entspringt, fehlt. Der 
Innenast ist beim 2 gestreckt, deutlich dreigliedrig, trigt am Ende eine 
lange Borste (Taf. 1, Fig. 21), beim @ ist er als Greiforgan entwickelt, 
das 3. Glied gegen das 2. einschlagbar (Taf. 1, Fig. 18, 19). 

Die Mandibel (Tafel 3, Fig. 6) ahnlich wie bei Cypridina, besonders 
die Kauplatte (Taf. 1, Fig. 20) von ahnlicher Gestalt, die Borsten am 
Vorderrand des 3. Tastergliedes stark vermehrt, die Driise erstreckt sich 
distal bis in die Basis des 3., proximal nur bis in die Mitte des 1. Tas- 
tergliedes. Bemerkenswerth ist ein dichter Besatz mit feinen Haar- 
borsten, welche in Gruppen von 3-8 beisammen stehen ; solche Gruppen 
finden sich am 1. Tasterglied, derber am Kaufortsatz (Tafel 1, Fig. 17, 
20). 1. Mazille (Tafel 3, Fig. 3) von ahnlichem Ban wie bei Oypridina, 
ebenso die 2. Mawille (1. thoracale Gliedmaasse, Tafel 3, Fig. 4; Tafel 2, 
Fig. 8, 9). Die Borsten des letzten Gliedes sind stark vermehrt, die 
zahnartigen Gebilde der einen Reihe sind viel kraftiger als die der 
anderen, die der kriiftigeren Reihe sind stark gekrimmt. Die 2. thoracale 
Gliedmaasse (Taf. 3, Fig. 7) ahnlich wie bei Cypridina, doch auffallend 
klein, mit thnlichen Gruppen von Haarborsten wie der Mandibular- 
taster. Putzfuss (Tafel 3, Fig. 5) ausserordentlich lang und wohl ent- 
wickelt, die Ringe sehr kurz, das letzte + mit sehr zahlreichen Borsten 
(ich schiitze jederseits ttber 200), welche simmtlich von typischen Bau ; 
das Ende mit 1 langen Reihe sehr zahlreicher kleiner Zahne, welcher 
Reihe nur ein einzelner kleiner Zahn gegeniiber steht (Tafel 1, Fig. 15). 
Furea mit 11 Dornenpaaren (die Zahl ist anscheinend constant). Die 
Dornen nehmen ziemlich regelmiissig von der Spitze nach der Basis zu an 
Groésse ab; das in Figur 15, Tafel 2 gezeichnete Zuriickbleiben des 5. 
Dornes findet sich nicht constant. 

Die Oberlippe (Tafel 1, Fig. 3, Ol.) besteht aus einem unpaaren, nach 
vorn gerichteten Kamm und einem breiten, queren Wulst, welcher direct 


eS ee 


MULLER: OSTRACODEN. 159 


vor dem Munde liegt ; die Miindungen der Driisen finden sich am vor- 
deren Rand des Kammes, sowie an den Seiten des queren Wulstes. Be- 
merkenswerth ist der Bau des Magens (Tafel 1, Fig. 10). Derselbe besteht 
aus einer inneren Epithelschicht ; die Epithelzellen waren stets sehr diinn 
und hoch, was unzweifelhaft eine Folge der starken Contraction war, 
dann folgt ein structurlose, stark gefaltete Membran, dieselbe stammt 
vermuthlich von den Epithelzellen. Nach aussen von ihr liegen zahl- 
reiche Muskelfasern (J//.), die sich anniéhernd unter rechtem Winkel 
kreuzen, ein dichtes Netzwerk bilden, es folgt eine Schicht von Zellen, 
die dicht mit braunschwarzem Pigment erfiillt sind (Leberzellen), 
schliesslich ein dichtes Netzwerk von Bindegewebszellen, deren Aus- 
laufer bis zum Riicken, in der Herzgegend bis zur Schale reichen; sie 
lassen den Magen wie mit einem dichten Pelz bekleidet erscheinen. 
Stets fand ich den Magen stark contrahirt und leer, augenscheinlich 
erfolgt im Netz oder bei der Conservirung stets eine starke Contrac- 
tion und Entleernng. Bei der anderen Art, wo der Magenwand die 
Muskelfasern fehlen, fand ich im Magen Reste einer Salpe. 

Sinnesorgane. — Paarige Augen. Dieselben erheben sich als kleine 
Warzen ziemlich frei ttber der Basis der 1. Antenne, hinter den Seiten- 
theilen des Frontalorgans (Tafel 1, Fig. 3, Oc.). In jedem Auge finden 
sich 4 langliche, etwa birnformige Blasen (die Zahl 4 scheint constant 
zu sein). Die Oberfliche der Blase ist glatt, auf der inneren Fliche 
der Wandung finden sich zahlreiche Rippen oder Leisten von stiirkerein 
Lichtbrechungsvermoégen, dieselben bilden ein Gewirr von ziemlich 
scharf begrenzten Linien, die einen unregelmissigen, meist geschliin- 
gelten Verlauf haben (Tafel 1, Fig. 14). Bei jiingeren Thieren ist die 
Anordnung der Rippen einfacher, auch zeigen sie bestimmte Bezie- 
hungen zu den zahlreichen Kernen, die sie meist im Bogen umziehen 
(Tafel 1, Fig. 11, da die Fig. 11 von Gigantocypris pellucida staramt, 
so ist es auch moglich, dass es sich um Artunterschiede handelt). Am 
proximalen, spitzen Ende geht die Blase in einen feinfasrigen Fortsatz 
itber, den Nerven. Ich betrachte diese Blasen als eigenthitmlich 
umgestaltete Rhabdome. Welche Function freilich diese Blasen haben, 
das ist schwer einzusehen. Ihre Verbindung mit einem Nerven spricht 
fiir ein Sinnesorgan, und hier scheint jede andere Deutung als die eines 
Sehorganes ausgeschlossen, ich komme auf die Frage noch einmal kurz 
zurick. Linsen habe ich in dem Auge vollstiindig vermisst, auch 
Pigment fehlte, doch kann es in Folge der Conservirung verloren gegan- 
gen sein; hei G. pellucida hatte die Blase ein bréunliche Farbung. 

Das Frontalorgan ragt als grosser, nasenformiger Kérper oberhalb des 


160 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


Ursprungs der 1 Antenne vor, im Profil zum Theil verdeckt durch den 
Stamm der 2 Antenne. Wir unterscheiden an demselben 3 in der Nase 
selbst liegende und 2 nahe der Nasenwurzel liegende K6érper von dun- 
klerer Farbung. Die Lage dieser K6rper wird durch Fig. 3, 5, Tafel 
1 veranschaulicht. Die in der Nase selbst liegende Gruppe besteht 
aus zwei grésseren diinnen, etwa senkrechten, nach unten divergirenden 
Platten (a) von annihernd dreieckiger Gestalt und einer kleineren, 
diinneren, liinglichen Platte (b), welche sich zwischen die horizontalen 
Rénder der verticalen Platten schiebt. Die Platten bestehen aus einer 
annihernd bomogenen Grundsubstanz, in der sich zahlreiche verzweigte 
Streifen einer dichteren, starker farbbaren Substanz finden (Tafel 1, 
Fig. 12), Die Streifen sind nicht scharf gegen die Grundsubstanz ab- 
geerenzt. Die Anordnung der Streifen lasst stellenweis ahnliche Bezie- 
hungen zu den Kernen erkennen wie beim Rhabdom (Tafel 1, Fig. 11). 

Bei der horizontalen kleinen Platte, von der die Figur 12 stammt, ist 
das Bild ein ziemlich tbersichtliches, in Folge der geringen Dicke der 
Platte liegen alle Streifen annihernd in einer Ebene, complicirter wird 
das Bild an den dickeren, verticalen Platten, bei denen die Zahl der 
dichteren Streifen eine gréssere ; bei den seitlichen Koérpern (Fig. 3, 
5 c), die einen etwa ovalen Querschnitt besitzen, ist sie naturgemass am 
gréssten ; die Oberflache derselben bietet das Bild einer sehr dichten, 
welligen Querstreifung (Tafel 1, Fig. 13). Auf Schnitten (Tafel 1, Fig. 4) 
zeigt sie sich in ganzem Umfang durchsetzt von dichteren, mit einander 
anastomosirenden Streifen. Die Innervirung erfolgt bei den seitlichen 
Korpern von der distalen Flache aus, bei den senkrechten Lamellen 
anscheinend von der iiusseren, bei der kleinen verticalen von der unteren 
Flache, doch habe ich bei den zuletztgenannten keine volle Klarheit 
erlangt.* Die Kerne liegen stets lediglich an derjenigen Fiche, 
oder nahe derjenigen Fliche, an welcher die Nerven an den Korper 
herantreten. Die beiden seitlichen Korper sind von einem braunlichen 
Pigment umhiillt, das einer sehr diinnen, structurlosen Membran aufliegt. 
Die Grenzen der Pigmentirung habe ich nicht sicher feststellen koOnnen. 
Der mittleren Gruppe scheint eine pigmentirte Hille zu fehlen, doch 
diirfte es sich auch hier um einen Verlust des Pigmentes in Folge der 
Conservirung handeln. 

Was die morphologische Deutung des Organs anbetrifft, so habe ich 
schon ausgesprochen, dass ich es als das Frontalorgan anspreche, und 

* Ich muss die Unbestimmtheit der Resultate mit dem geringen Material, auf das 


ich angewiesen war, entschuldigen. Nur das eine wohl erhaltene ¢& lieferte fiir die 
Untersuchung des Frontalorgans brauchbare Resultate. 


MULLER: OSTRACODEN. 161 


zwar halte ich die mittlere Gruppe fiir homolog dem unteren Abschnitt 
anderer Cypridiniden, die seitlichen K6rper den oberen paarigen Ab- 
schnitten. An Stelle der Gruppen von Sehstibchen sind solide Kérper 
von ihnlicher Zusammensetzung getreten. Bei den seitlichen Kérpern 
(c) erkennt man ohne Weiteres die Ubereinstimmung in der Art der 
Innervirung und Lage der Kerne. Wie bei den tibrigen Cypridiniden 
haben wir dichtere und stirker lichtbrechende Stibchen, welche einer 
homogenen Masse eingebettet sind, an der man hiufig Zellgrenzen nicht 
zu erkennen vermag. Der wesentliche Unterschied wiirde in der 
Beschaffenheit der Stibchen liegen, die hier viel linger und wellig 
- gebogen sind, sich verzweigen, mit einander anastomosiren. Weniger 
klar liegen die Verhaltnisse bei der mittleren Gruppe. Wir miissen 
annehmen, dass der urspriinglich einfache Abschnitt zunichst in drei 
kleinere Korper zerfallen ist. Bei der flachenhaften Entwicklung dieser 
K6rper mussten die Sehstiibchen ihre Lage iindern, sie entwickelten sich 
parallel der Oberflaiche der Korper. Der zitzen oder stabformige Fort- 
satz des Frontalorgans fehlt ; nur eine seinem Basalstiick entsprechende 
Anhiiufung von Kernen am unteren Rand der Nase und an der Nasen- 
spitze ist als Rest desselben anfzufassen. 

Das Herz ist auffallend zart und diinnhdutig, so dass es bei der Pri- 
paration des Thieres vollstindig zusammenfiallt. Der ausserordentlichen 
Vermehrung der Leibeshohlenfliissigkeit entspricht also nicht eine Ver- 
stiirkung des Circulationsapparates, im Gegentheil ; ich vermuthe dass 
das Herz kaum noch functionirt. Die mannlichen Geschlechtsorgane 
schliessen sich im Bau eng. denen anderer Cypridiniden an, die weib- 
lichen Geschlechtsorgane konnte ich nicht genauer untersuchen, da 
simmtliche Q stark verdriickt waren; die Eier werden in grosser Zahl 
im Brutraum getragen. Der Schliessmuskel ist sehr schwach im Ver- 
haltniss zur Grosse des Thieres, die Zahl der Muskelbiindel ist gering, 
die Biindel selbst sind schwach, doch zeigen sie deutliche Querstreifung. 

Versuchen wir aus der Organisation des Thieres uns eine Vorstellung 
seiner Lebensweise zu bilden! Schon Eingangs sprach ich die Vermu- 
thung aus, dass die Art ausschliesslich frei schwimmend lebt, sich nicht 
am Grunde bewegt. Fiir die Mehrzahl der Cypridiniden lehrt die Beo- 
bachtung des lebenden Thieres, dass dieselben vorwiegend an den Grund 
gebunden sind, aber auch fiir die Halocypriden, die man gewohnlich als 
rein pelagische Formen betrachtet, habe ich die Ansicht ausgesprochen, 
dass sie sich wenigstens zeitweis am Grund aufhalten. Die Griinde, 
die ich fiir diese Ansicht an anderem Ort (Monographie der Ostracoden, 
p- 13) geltend machte, waren im wesentlichen folgende: 1. das hohe 


162 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


specifische Gewicht, besonders bedingt durch die Verkalkung der 
Schale ; 2. die Koérperform, der Mangel flichenhafter Ausbreitung, die 
ein freies Schweben ganz ohne oder mit nur geringem Kraftaufwand 
nicht gestattet ; 3. der Bau der Gliedmaassen, welche zum Theil zum 
Anklammern geeignet sind. Wie verhalt sich in dieser Beziehung 
Gigantocypris 2? Wie gesagt, fehlt der Schale jede Spur von Kalkablage- 
rung, zudem tritt die feste Substanz der Leibeshdhlenflissigkeit gegen- 
iiber sehr zuriick, das specifische Gewicht kann kaum hoher als das des 
umgebenden Mediums sein. Flachenhafte Ausbreitungen fehlen so gut 
wie bei anderen Ostracoden, doch ist, wie gesagt, der Umfang im Ver- 
hiiltniss zur Kérpermasse ein sehr grosser, so dass man sich sehr wohl 
denken kann, dass das Thier ohne Zuhilfenahme seiner Ruder wie eine 
leichte Blase durch das Wasser treibt, ohne unterzusinken. 

Von besonderem Interesse ist eine Betrachtung der Gliedmaasen. Die 
Gliedmaassen aller Cypridiniden sind ungeeignet zum Kriechen und 
Anklammern, und das gilt auch von Gigantocypris, wohl aber vermégen 
die Mehrzahl der Cypridiniden sich in den Grund einzugraben, die 
Gliedmaassen zeigen mancherlei Anpassung an diese Lebensweise, 
welche Anpassungen allerdings gerade bei der Gigantocypris am niachsten 
verwandten Gattung Cypridina am wenigstens ausgebildet sind. Wie 
verhiilt sich in dieser Bezichung Gigantocypris ? Die 1. Antenne bethei- 
ligt sich bei Cypridina am Kingraben, bei G. ist sie viel schlanker, zum 
Eingraben weniger geeignet, besondere als Anpassungen an das Ein- 
graben aufzufassende Eigenthiimlichkeiten fehlen beiden Gattungen. 
2, Antenne: Die Borste des 2. Schwimmastgliedes ist bei Cypridina 
kurz, ungefiedert, bedornt, bei Gigantocypris ebenfalls kurz, ungefiedert, 
aber unbedornt. Der Dorn, der bei Cypridina neben jeder Schwimm- 
borste entspringt, und vermuthlich ebenfalls dem Eingraben dient, 
fehlt. Der Mandibulartaster betheiligt sich ebenfalls am Eingraben, 
er ist bei Gigantocypris, vergleichen mit der Maxille, verhialtnissmissig 
kiirzer als bei Cypridina, tibrigens wohl entwickelt, was sich aus 
seinem Antheil an der Nahrungsaufnahme erklirt. Eine hervorragende 
Rolle spielt die Furca beim Eingraben, sie ist bei Gigantocypris auffal- 
lend kurz und schwach entwickelt, besonders sind die Dornen diinn und , 
zerbrechlich. Wird die Furca nicht mehr zum Eingraben verwendet, so 
erscheint eine Gliedmasse, bestimmt die Furca abzubiirsten ziemlich 
iiberfliissig, und in der That ist die 2. thoracale Gliedmaasse (Tafel 3, 
Fig. 7) ziemlich schwach entwickelt, wiihrend der Putzfuss eine ausser- 
ordentliche Linge erreicht, entsprechend der grossen Fliche, die er zu 


reinigen hat. 


MULLER: OSTRACODEN. 163 


Uebrigens bedarf es kaum dieser Thatsachen, um den Beweis zu 
liefern, dass sich Gigantocypris nicht einzugraben vermag ; der Umfang 
und die Zartheit der Schale allein wiirden eine iihnliche Bewegung 
unmdglich machen. Ich glaube nach allem Gesagten, dass sich Giganto- 
cypris ausschliesslich frei schwimmend bewegt, nach meiner Auffassung 
wiirde es der einzige bis jezt bekannt gewordene Ostracode mit dieser 
Lebensweise sein. 

Mit der Frage nach der Art der Bewegung berithrt sich eng eine 
andere, die, nach der Modglichkeit, die Schale zu 6ffnen. Nach der 
Darstellung, die ich oben vom Bau der Schale gegeben habe, driingt 
sich die Vorstellung auf, dass das Thier die Schale gar nicht zu 6ffnen 
vermag, das wiirde schon daraus folgen, dass beide Schalenhilften fiir 
% ihrer Peripherie fest mit einander verbunden sind. Auch die geringe 
Entwicklung des Schliessmuskels scheint diese Annahme zu befiirworten. 
Man konnte sich wohl vorstellen, dass nur die 1. und 2. Antenne durch 
die Rostralincisur herausgestreckt werden kénuten, dass sich, abgesehen 
vom Umherschwimmen, die Thatigkeit des Thieres darauf beschrinkte, 
durch die Bewegung der 1. thoracalen Gliedmaasse (2. Maxille) einen 
Wasserstrom zu erzeugen, der bei geschlossener Schale durch die Rostral- 
incisur ein, durch die hintere Oeffnung austriite und sowohl die Ath- 
mung vermitteln als auch die Nahrung zufithren wiirde. Im allgemeinen 
mag sich in der That das Leben der Thiere in dieser Weise abspieclen, 
doch sprechen einige Griinde dafiir, dass doch ein gewisses Oeffnen der 
Schale erfolgen kann ; so wire es unmdéglich, die Beute, die ich ziemlich 
unzerkleinert im Magen von'G. pellucida fand, durch die Rostralincisur 
allein in den Schalenraum und zum Mund zu bringen; weiter wire eine 
Begattung, ein Vorstrecken des Penis ohne Oeffnen der Schale unmoglich, 
Unzweifelhaft ist aber der Mechanismus beim Oeffnen der Schale ein 
wesentlich anderer, es koénuen nicht beide Schalenhilften als Ganzes von 
einander entfernt, sondern es kénnen nur die Rinder auseinandergebogen 
werden, wahrend die iibrige Schale nur zum kleineren Theil in Mitleiden- 
schaft gezogen wird ; das witrde ja auch bei der Weichheit und Biegsam- 
keit der Schale sehr wohl méglich sein. Immerhin scheint ein solches 
Oeffnen den postoralen Gliedmaassen und der Furca nur wenig Spielraum 
zur Bewegung zu lassen. 

Noch bleibt die Frage zu erdrtern, in welcher Bezichung die sehr auffiil- 
lige Umgestaltung der Sehorgane zur Lebensweise steht? Da die 
gesammelten Thiere z. Th. aus bedeutender Tiefe stammen, so liegt der 
Gedanke nah, dass wir in der eigenthitmlichen Form eine Anpassung an 
das Leben in grossér Tiefe zu sehen haben; vielleicht sind diese merk- 


164 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


wiirdigen Augen befihigt, noch bei sehr schwachem Licht Differenzen in 
der Helligkeit wahrzunehmen. Ich lasse noch eine kurze Gattungs- 
diagnose, sowie eine Differentialdiagnose beider Arten folgen. 


Gigantocypris, nov. gen. 


Schale diinn und zart, nicht verkalkt, beide Schalenhalften nur an- 
deutlich gegen einander abgegrenzt, fiir etwa 3 der Peripherie mit einan- 
der verschmolzen, mit kleiner, aber deutlicher Rostralincisur. K6rper 
stark aufgetrieben. 1. Antenne gestreckt, 7 gliedrig, Sinnesborste des 
5. Gliedes schwach gefiedert, in beiden Geschlechtern gleich ; beim ¢ 2 
Borsten des letzten Gliedes mit Haftorganen; 2 Antenne: die erste 
Borste des Schwimmastes kurz, ungefiedert, unbedornt. Innenast drei- 
gliedrig, beim ¢ als Greiforgan functionirend, das letzte Glied ein- 
schlagbar, Mandibel mit einfachem, einspitzigem, riickwiirts gerichtetem 
Fortsatz des Basalgliedes und wohl entwickeltem 4 gliedrigem Taster, 1. 
Maxille von typischem Bau, mit 3 Kaufortsaitzen und 3 gliedrigem Taster. 
1. thoracale Gliedmaasse (2. Maxille) mit 2 Reihen zahnartiger Borsten. 
2. thoracale Gliedmaasse deutlich 4 gliedrig, mit schwacher Musculatur. 
Putzfuss sehr lang, mit sehr zahlreichen Borsten und einer Reihe kleiner 
Zaibne. Furca ohne deutliche Differenzirung in Haupt und Nebendor- 
nen. Oberlippe umfangreich, ohne zitzenartige Fortsiatze, die Mehrzahl 
der Driisen miindet auf einer Kante am Vorderrand. Frontalorgan sehr 
umfangreich, die oberen Gruppen weit von der mittleren getrennt, die 
mittlere in 3 Korper aufgeloést, ohne zitzen oder stabformigen Fortsatz. 
Paarige Augen vorhanden (in beiden Geschlechtern 1), mit 4 eigenthiim- 
lich umgestalteten, blasenformigen Elementen, ohne Linsen. 

Wie schon hervorgehoben, schliesst sich Gigantocypris der Gattung 
Cypridina eng an, zeigt eine weitgehende Uebereinstimmung im Bau der 
Gliedmaassen. Als unterscheidende Merkmale waren hervorzuheben : 
der Bau der Schale, der Oberlippe und des Frontalorganes. Die oben 
gegebene Beschreibung bezog sich auf Gigantocypris Agassiz, neben 
dieser durch 5 Individuen (4 9,1 ¢) vertretenen Art fand sich noch ein 
Individuum einer zweiten Art; obwohl nur ein noch nicht geschlechts- 
reifes ¢ vorlag, habe ich doch geglaubt, bei dem besonderen Interesse, 
welches sich an die Gattung kniipft, die Art beschreiben zu sollen, zumal 
scharfe unterscheidende Merkmale existiren. 


Gigantocypris pellucida, n. sp. 


Schale, Gliedmaassen und sonstiger Kérperbau wie bei Agassiz ; am 
Putzfuss war die Zahl der Zibne in der Reihe an der Spitze viel kleiner 


MULLER: OSTRACODEN. 165 


(7 anstatt etwa 60; Tafel 1, Fig. 16,15). Neben den typischen Borsten 
(Tafel 1, Fig. 23), existiren solche mit einfachem pinselartigem Ende 
(Fig. 22), letztere sind viel seltner als die erstgenannten. In der 
Magenwand fehlen die Muskelfasern, an ihrer Stelle finden sich nur 
diinne, anscheinend nicht contractile Fasern, entsprechend ist der Darm 
an conservirtem Material nicht contrahirt ; ferner fehlen die zur Leibes- 
wand verlaufenden Bindegewebsfasern. Das untersuchte Thier stand 
vor der zur Geschlechtsreife fiihrenden Hiutung, es mass 16 mm., 
danach diirfte die Art etwas kleiner sein als Agassiz. 
Vorkommen: Si&immtliche Thiere sind gefischt nahe der Westkiiste 
von Central Amerika, niimlich : — 
0° 36’ n. Br. 82° 45’ w. 
0° 54’ n. Br. 91° 9! w. 
go 34 n..Brs 92° 6! w. 
26° 48’ n. Br. 110° 45’ w. 
Moe te br ATL> <0" w: 
in Tiefen von etwa 1700 Faden bis 100 Faden (offenes Netz). 
Vermuthlich bezieht sich auch die folgende Angabe* auf eine Art der 
Gattung Gigantocypris: Der Challenger brachte zwischen den Prinz 
Edwards und Crozet Inseln aus einer Tiefe von 1375 oder 1600 Faden 
mit dem Schleppnetz einen Ostracoden herauf, dessen weiche, skulptirte 
Schale eine Lange von 25 mm. und eine Hohe von 16 mm. hatte. Der 
Deckel (?) allein ist 3 mm. lang. Wahrscheinlich gehdrt dieser Ostra- 
code, von dessen K6rper nur der Kopf erhalten ist, zu keiner der bis 
jezt bekannten Familien. Die Angabe, dass nur der Kopf erhalten sei, 
diirfte sich vielleicht aus der geringen Grésse des KGrpers, vergleichen 
mit der Schale, erklaren. Leider fehlen nihere Angaben iiber das Thier, 
in den Challengerostracoden ist es nicht erwihnt. 


Sees 


HALOCYPRID A. 


Gesammelt sind 4 Arten Conchecia (Conchecissa) armata Cls., 1 
9 gefangen unter 10° 14’ n. Br. 96° 28’ w. L. in 100 Faden Tiefe. 
Ferner je 1 Q von 2 unbeschriebenen Arten; da der Fund zu einer 
scharfen Characterisirung der Arten nicht geniigt, unterlasse ich es, die 
Arten zu beschreiben. Schliesslich fand sich ein reichliches Material 
einer sehr stattlichen Conchecia : 


* Briefe von der Challengerexpedition von R. vy. Willemoes—Suhm. Zeitschrift 
wissensch. Zoologie, Bd. 24, p. XIII. 


166 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


Concheecia Agassizii, n. sp. 


(Tafel 2, Fig. 1-7, 12-14, 16-18.) 


Schale sehr derb, ungewohnlich derb und widerstandsfahig fiir einen 
Halocypriden, besonders fiir eine Conchecia, indessen im Verhiltniss 
zum Korper zu klein, so dass sie den K6rper nicht ganz umhiillt, vielmehr 
stark klafft. Die des ? nicht ganz noch einmal so lang wie hoch 
(Hohe zur Linge etwa 1:1,9), am breitesten etwa auf 2 der Linge, von 
wo sich die Schale stark nach vorn verschmiilert, hintere untere Ecke breit 
gerundet, der Hinterrand gerade, bildet mit dem Dorsalrand einen spitzen 
Winkel mit abgerundeter Spitze. Rechte und linke obere Ecke nicht 
dentlich verschieden. Schale des ¢ etwas gestreckter, etwas iiber noch 
ein mal so lang wie hoch, nach vorn weniger stark verschmalert ; der 
Winkel, welchen Dorsal- und Hinterrand mit einander bilden, grésser 
als beim 9, doch immer noch kleiner als ein rechter, der Rostralfort- 
satz des @ nur wenig stirker in die Héhe gebogen als beim 9, mit 
kurzer, abgesetzter Spitze. In beiden Geschlechtern zeigt die Schale 
eine schwach entwickelte, nur schwer nachweisbare Skulptur ; dieselbe 
besteht aus zahlreichen undeutlichen Linien, welche in der vorderen 
Halfte eine parallele Streifung oder langgestreckte Rhomben, in der 
hinteren Hilfte eine polygonale Felderung bilden. Die Streifen ver- 
laufen anniaihernd senkrecht zur Riickenlinie. Die unsymmetrischen 
Driisengruppen sind wohl entwickelt, die Ménnchendriisen habe ich 
nicht auffinden kénnen. 

1. Antenne des ¢: die Hauptborste sehr lang, iber 3 mal so lang als 
die 1. Antenne, mit sehr zahlreichen riickwirts gerichteten Spitzen, ich 
zihle gegen 100 Paare; die Paare stehen sehr dicht und regelmassig 
neben einander, so dass die eine Spitze die benachbarte im Profil ver- 
deckt, folgen sich sehr dicht, veriindern proximalwarts Gestalt und 
Anordnung nicht oder nur unbedeutend ; auf die riickwiirts gerichteten 
Spitzen folgen distal noch einige schwiichere, vorwirts gebogene borsten- 
artige Anhiinge (Tafel 2, Fig. 18). Die zwei schwiacheren Borsten sind 
annihernd gleich lang, etwas liinger als die halbe Hauptborste, sie 
tragen einzelne vorwiirtsgerichtete Spitzen. Von den beiden Sinnes- 
borsten erreicht die distale eine Linge von mehr als 3 der 1. Antenne, sie 
ist sonst in der Gattung Conchecia durchweg viel kiirzer. Beim @ ist 
die 1. Antenne viel schwiicher als beim ¢, zeigt aber eine wohl ent- 
wickelte Muskulatur, die Hauptborste erreicht die 2—3fache Linge der 
1. Antenne, die 4 gleichlangen Sinnesschlaiuche etwa die Linge der 2 
ersten Glieder. 


MULLER: OSTRACODEN. 167 


Nebenast der 2. Antenne des 6 (Tafel 2, Fig. 3-5, 7): das Basal- 
glied von typischer Form, die eine Borste diinn gefiedert, das 2. Glied 
trigt ausser den 2 starken endstindigen Borsten gegeniiber dem Ur- 
sprung des letzten Gliedes zwei schwache, schlanke Borsten, von den 
beiden endstindigen Borsten erreicht die eine eine ausserordentliche 
Lange, ist etwa doppelt so lang wie der Aussenast mit seinen Schwimm- 
borsten, die andere erreicht nicht ganz die halbe Liinge der ersten. Am 
letzten hakenartig gebogenen Glied bleiben die 3 Borsten sehr kurz, 
erreichen etwa nur } der lingsten Borste des Innenastes, der Haken ist 
auf beiden Seiten stark gebogen, in eine Spitze ausgezogen, rechts und 
links in der Gesammtform nicht sehr verschieden ; auf der einen Seite 
zeigt er nahe der Basis 2 einander gegeniiberstehende zahnartige Vor- 
sprunge. 

Beim 9 (Tafel 2, Fig. 6, 13) ist der Nebenast kiirzer, die langste 
Borste erreicht noch nicht 2 der Linge wie beim @, die Borsten des 
letzten Gliedes bleiben wie beim @ sehr kurz, an Stelle der 2 itberziih- 
ligen Borsten des vorletzten Gliedes beim @ findet sich nur eine. Beide 
Borsten des 1. Gliedes sind ungefiedert. 

Frontalorgan mit einfachem, erweitertem Endstiick, dessen Form nicht 
besonders charakteristisch ; das des 9 dem des @ fnhlich, nicht ganz 
noch einmal so lang als die 1. Antenne, das erweiterte, nicht beweglich 
abgesetzte, stark bedornte Endstiick schlanker als beim 2. 

Das Thier erreicht eine Grésse von 4,8 mm. 

Gefischt im Golf von Californien in einer Tiefe von etwa 700 Meter 
(offenes Netz). 


168 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


FIGURENERKLAERUNG. 
a. L. Aeussere Lamelle. N. Nerv. 
An,, Ang. Erste, zweite Antenne. Oc. paariges Auge. 
C. Herz. Oe. CEsophagus. 
Dz. Darm. Ol. Oberlippe. 
Dr. Driisenmiindung. Pe. Penis. 
F. Furca. Pf. Putzfuss. 
Ff. Furcalfeld. R. Rand. . 
Fr. Frontalorgan. R,. innere Randleiste. 
G. Gehirn. S. Saum. 
Bl Dp innere Lamelle. T. Hoden. 
Tr. Innenrand. Vi. Verwachsungslinie. 
Mi. Muskel. 

TAFEL 1. 


Fig. 1-5. Gigantocypris Agassizii. 

Fig. 1,2. @, ganzes Thier, von der Seite und von unten. 3 X 

Fig. 3. Ventrale Koérperhilfte nach Entfernung der Gliedmaasen. 7 X. Die 
Buchstaben a, b, ce, bezeichnen die gleichen Theile wie in Fig. 5. 

Fig. 4. Schnitt durch einen seitlichen Kérper (c) des Frontalorganes. 100 X 

Fig. 5. Frontalorgan von vorn. 7 X (a, b, c vergl. Fig. 3). 

Fig. 6,7. Gigantocypris pellucida @ juv. Rostralincisur und hinteres Ende des 
Schalenschlitzes von vorn, resp. unten. 7 X 

Fig. 8-10. Gigantocypris Agassizii. 

Fig. 8,9. @ Rostralincisur und hinteres Ende des Schlitzes von innen. 8 X 

Fig. 10. 9 Schnitt durch die Magenwand. 162 X 

Fig. 11. Gigantocypris pellucida. Einzelnes Rhabdom. 100 X 

Fig. 12-15. Gigantocypris Agassizii. 

Fig. 12. @ Distales Ende des Kérpers b des Frontalorganes von der Flache. 
100 x 

Fig. 13. Q Korper ec. des Frontalorganes. 380 X 

Fig. 14. & Stiick eines Rhabdoms. 162 X 

Fig. 15. Q Spitze des Putzfusses. 100 X 

Fig. 16.  Gigantocypris pellucida.  juv. Spitze des Putzfusses. 100 X 

Fig. 17-21. Gigantocypris Agassizit. 

Fig.17. @ Stiick vom 1. Tasterglied der Mandibel. 66 X 

Fig. 18, 19. @ Spitze des Innenastes der 2. Antenne, 162 X, und Innenast. 80 

Fig. 20. Q Kaufortsatz der Mandibel. 66 X 

Fig. 21. 9 Innenast der 2. Antenne. 30 X 


Fig. 


MULLER: OSTRACODEN. 169 


22,23. Gigantocypris pellucida. @ juv. Spitzen von 2. Borsten des Putz- 
fusses. 400 X 


.24.  Gigantocypris Agassizii. @ 2 letzten Glieder der 1. Antenne mit einem 


Theil der Borsten (weggelassen sind die 2 Sinnesborsten und eine 
starke Borste des letzten Gliedes). 66 X 


7 TAFEL 2. 
‘Fig. 1-7. Conchecia Agassizii. 
Fig. 1,2. Schale im Profill ¢,29;19x 
Fig. 3. & Innenast der 2. Antenne mit der Basis der Borsten, von innen, unter 
Deckglas. 100 x 
Fig. 4 & 2 Letzten Glieder des rechten Innenastes. 100 X 
Fig. 5 & Rechter Innenast. 26 X 
Fig. 6. Q Innenast, frei liegend, sonst wie 8. 100 
Fig. 7. & Rechter Innenast, frei liegend, sonst wie 3. 100 X 
Fig. 8-10. Gigantocypris Agassizii. 
Fig. 8,9. Kautheil der 1. thoracalen Gliedmasse (2. Maxille) von innen, 26 X und 
Zahne beider Zahnreihen. 100 « 
Fig. 10. Erster Furcaldorn von aussen. 50 X 
Fig. 11. Gigantocypris pellucida. & juv. Furca und Anlage des Penis von vorn. 
(es 
Fig. 12-14. Conchacia Agassizii. 
Fig. 12. 9 Kaufortsatz der Mandibel von innen, am Taster haftend. 216 x. 
Fig. 13. 2 Innenast der 2. Antenne. 26 X 
Fig. 14. 9 1. Antenne und Frontalorgan. 66 
Fig. 15. = Gigantocypris Agassizii 9 Furca (nur ein Ast ist gezeichnet). 19 x 


. 16-18. Conchaecia Agassizi @. 
. 16. 1. Antenne und Frontalorgan, rechte Antenne entfernt. 66 X 
. 17,18. Bezahntes Stiick einer Nebenborste und Ende der Zahnreihe der 


Hauptborste der 1. Antenne, beides 400 X 


TAFEL 3. 


Simtliche Figuren stellen Gliedmaassen eines geschlechtsreifen 9 von Giganto- 
cypris Agassizii von 23 mm. Schalenlinge dar; alle Figuren 10 X vergrossert. 


Fig. 
Fig. 
Fig. 
Fig. 
Fig. 
Fig. 
Fig. 


1. 2. Antenne. 

2. 1. Antenne. 

3. 1. Maxille. 

4, 1. thoracale Gliedmaasse (2. Maxille). 
5. Putzfuss. 

6. Mandibel. 

7. 2. thoracale Gliedmaasse. 


VOL. XXVII. — NO. 5. 2 


Me) Oe ap 
Peay 
pies 


i ALBATROSSEX16010STRACODS. 


5 Ae Ls = : @ i 


oy 


sa 


E. Crisana, lith. New Haven, Ct. 


ALBATROSS EX.1891.0STRACODS. 


PLATE II. 


8 4 
i 


MeiA AVT AT | 


tH) 
(ese ATUL 


mt 


E Crisand, lith., New Haven, Ct 


PLATE TIL 


E. Crisand, lith:, New Haven, Ct. 


w) 


S EX.1891.0STRACODS. 


ALBATROS 
.G.H.Miller, del. 


aie eee 


of the Museum of Comparative Zoology 
AT HARVARD COLLEGE. 
Vou. XXVII. No. 6. 


STUDIES IN MORPHOGENESIS. 


IV. 


IN ONTOGENY. 


By C. B. DavENpPoRT. 


CAMBRIDGE, MASS., U.S. A.: 
PRINTED FOR THE MUSEUM. 


NovemBer, 1895. 


NOV 21 1806 


Bulletin of the Museum of Comparative Zoology 
AT HARVARD COLLEGE. 
Vou: 2X Vil. -No. 6: 


STUDIES IN MORPHOGENESIS. 


Ve 


PRELIMINARY CATALOGUE OF THE PROCESSES CONCERNED 
IN ONTOGENY. 


By C. B. Davenport. 


CAMBRIDGE, MASS., U.S. A.: 
PRINTED FOR THE MUSEUM. 


Novemeer, 1895. 


No. 6.— Studies in Morphogenesis. —1V. A Preliminary Cat- 
alogue of the Processes concerned in Ontogeny.» By C. B. 
DAVENPORT. 


CONTENTS. 

4 Pace 
@eroauction: Limitations of the Paper . ... . . . « » « « © « » « A738 
geeceneral Ontogenetic Processes . . . . - « © «© «© « «© «© «© « « « LTA 
B. Special Ontogenetic Processes . . . 174 

I. Ontogenetic Processes occurring in Migeatory Pentoplasiiie Bodice 
(Mesenchyme) .... . . 175 

II. Ontogenetic Processes occurring in Blanvated Protoplaanie Boies 
(Fibres, Threads, Cords, Tubules) . . .. . a ows, teh 
III. Ontogenetie Processes occurring in Protoplasmic Tae BD came ge ton: 
IV. Ontogenetic Processes occurring in Protoplasmic Masses. . . . 192 
Recapitulation and General Remarks. . . . . . . . 1... « « « 19 


Most important perhaps of all the problems which the biologist sees 
lying unsolved before him is that of the development of the individual, 
—a problem to which, from the time of Aristotle, zodlogists have re- 
peatedly turned, although scarcely hoping for its eventual solution. 

Without attempting to consider the various theories of Ontomorpho- 
genesis which have at different times been offered, it is sufficient to state 
that it is now generally agreed that ontogenesis is a process, or rather a 
complex of processes, taking place in the protoplasm of the developing 
individual. 

Now it is a highly probable belief that no movement takes place in 
protoplasm except as a response to stimuli. The very fact that onto- 
genesis is a complex of actions indicates that there must be a large 
number of stimuli raining in upon the different parts of the developing 
protoplasm to which they respond. 

In order to gain some idea of what the stimuli are, it is first neces- 
sary to analyze the ontogenetic complex of processes into its simple 
elementary ones. 

It is the aim of this paper to make such an analysis into the elemen- 
tary ontogenetic processes as a basis for determining the nature of the 
exciting stimuli. 

1 Contributions from the Zodlogical Laboratory of the Museum of Comparative 


Zoblogy at Harvard College, E. L. Mark, Director, No. L. 
VOL. XXVII. — NO. 6. 1 


174 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY, 


Other authors have devoted chapters to “ Developmental Processes,” 
but none of these can be considered as at all complete. 

Thus O. Hertwig, in his “ Text-Book of the Embryology of Man and 
Mammals” (English Translation by E. L. Mark, 1892, p. 76), has a 
“General Discussion of the Principles of Development.” He recog- 
nized two main ones: (1) the principle of unequal growth (producing 
folds which are either invaginations or evaginations, and which may unite 
along their edges) ; and (2) the principle of histological differentiation. 

Minot, in his Human Embryology, in a chapter on “ Differentiation,” 
seems to think also that these two processes are sufficient to explain the 
differentiation of organs. 

More important in this connection than either of the preceding is the 
recent paper of Herbst in the ‘‘ Biologisches Centralblatt ”’ (Vol. XIV. 
Nos. 18-22). This author, after reviewing the literature upom taxism 
and tropism, explains as phenomena of the same order certain ontogenetic 
processes. He has not, however, attempted to catalogue all the onto- 
genetic processes. 


Before beginning the present catalogue, I may state that I distinguish 
between ontogenetic principles and ontogenetic processes. Under the 
first head I include such laws of development as terminal growth, repeti- 
tion of parts and bilateral symmetry in development. These I have not 
attempted to catalogue. The present paper is concerned only with the 
latter group, which comprises the different elementary operations or ac- 
tions exhibited in ontogeny. 

These may be divided into two classes: 1. the grosser ontogenetic pro 
cesses; and 2. histogenic processes. This paper deals with the first 
class only. 

In discussing the grosser ontogenetic processes we may distinguish 
(A) those of a more general nature from (B) the more special ones. 

A. The general ontogenetic processes comprise those of a general physio- 
logical character. Of these, at least three are commonly recognized :— 

1. Growth (including both the results of assimilation and of imbibition 
of water). 

2. Nuclear division. 

3. Secretion. 

B. The special ontogenetic processes are differential in character, i. e. 
the differentiation of the body is effected by them. 

These processes may be classified, first of all, according to the form of 


DAVENPORT: PROCESSES CONCERNED IN ONTOGENY. Lia 


the protoplasmic bodies in which they occur. These exist either as 
(I.) isolated cells, or as larger multinucleated bodies. Of the latter we 
may recognize three classes: (II.) bodies extended chiefly in one direc- 
tion, — threads, fibres, tubules; (III.) those which extend as a layer; 
and (IV.) those in which the three dimensions are more nearly equal, 
forming solid masses. 

I propose now to discuss the processes occurring in each of these four 
classes." 


I. ONTOGENETIC PROCESSES OCCURRING IN MiGrRatoryY PROTOPLASMIC 
Bop1es — MESENCHYME.? 


1. Migration of Nodal Thickenings in a Protoplasmie Mesh-work. This 
process is found, for example, in 
many Arthropod egys before the for- 
mation of the peripheral blastoderm. 
(Figure 1.) No one can doubt that 
protoplasm extends throughout the 
whole egg in the form ot a mesh- 
or foam-work, whose interspaces are 
filled with yolk. The protoplasm 
is aggregated around the nuclei at 
certain nodal points, which later 
migrate to the surface or through 
the yolk as vitellophags. Cf. K. 
& H.,® Figs. 7, 363, 417, 448, 472, Rimat 
473, 771. 


1 It may be a cause of dissatisfaction to some that this classification is not 
“strictly dichotomous”; it is still more serious that the different heads are not of 
co-ordinate rank or mutually exclusive. Of course, the classification employed in 
this list cannot be regarded as a final one. I hope, however, that I have succeeded 
in an attempt roughly to arrange the different items in a logical fashion. 

2 In the present paper the word “mesenchyme” is used as a name for all amoe- 
boid, migrating cells, of whatever origin. 

8 Throughout this paper certain abbreviations are used in referring to the books 
from which the figures are copied. These are: K. & TH. for Korschelt und [eider’s 
“Entwicklungsgeschichte ”; M. for Minot’s “ Human Embryology ”; and I.-M. for 
Hertwig’s “ Text-Book of Embryology of Man and Mammals,” translated by Mark. 


Fig. 1. Section through an egg of a Myriapod (Geophilus), showing the nodal 
thickenings (n. t.) in the act of migrating towards ti.e periphery of the egg. See 
K. & I, Fig. 449. 


176 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


2. Free Migration of Ameboid Bodies. This process differs from 
the preceding in that the migrating bodies are not connected together. 
It is characteristic of mesenchyme.Y I know that Dreyer (92, Jena. 
Zeitschr., XXVI. 359) and Sedgwick (94, Quart. Jour. Micr. Sci., 
XXXVII.) insist that the cells of mesenchyme, which are usually con- 
sidered unconnected like so many amcebz, are really nodal thickenings 
in an extensive mesh-work or foam-work, the intervening fluids being the, 
in some places confluent, vacuoles. Wherever mesenchyme has this 
structure, its migrations belong to the preceding class. But I believe 
there still remains a considerable residuum of cases falling under this 
head. 

This process is capable of division into two subprocesses; viz. (a@) mi- 
erating of mesenchyme out of a protoplasmic layer in order to become 
free, and (6) migrating through fluid-filled spaces. Both these processes 
are illustrated in Figure 2. Further illustrations will be found in K. & 
H., Figs. 102, 103, 170, 175-180, 
182, 186, 188-190, 207, 285, 559, 
596-598, 628, 698, 733, 809, and 
M., Figs. 121, 234, 239. 

The migratory processes named 
below are of subordinate rank to 
Nos. land 2. But, being fairly well 
marked and of considerable impor- 
tance, it is convenient to treat them 


as co-ordinate. 
Fic. 2. We may distinguish, first, move- 
ments of mesenchymatous elements 
towards and from each other, and, secondly, movements with reference to 
other protoplasmic masses. 

3. First among the former we recognize the aggregation of migratory 
protoplusmic bodies, and here we may distinguish three sub-classes accord- 
ing to the form of the resulting hody. 

a. First we have the aggregation of mesenchyme into a body with a 
chiefly linear dimension, — the formation of a thread, cord, or tubule. 

Examples of this process in Invertebrates are seen in the formation of 
the kidney of Lamellibranchs, which seems to be laid down as a cord-like 
aggregation of mesenchyme, and in that of the thread of the yolk glands 


Fig. 2. Section of Holothurian larva showing mesenchyme migrating out of a 
layer at a, and through a fluid-filled space at 6. From H.-M., Fig. 109. 


DAVENPORT: PROCESSES CONCERNED IN ONTOGENY. Laz. 


of Turbellaria according to Iijima (Zeitschr. f. wiss. Zool., XL. 455). 
Among Vertebrates, we have the observations of Paterson, (Figure 3,) 
according to which the sympathetic nerve arises by the aggregation of 
mesenchymatous elements into a strand; of His, who affirms the origin 
of the spinal and the olfactory ganglia from migrating cells; and of 
various authors, who make blood capillaries and lymph vessels arise by 
this process (cf. M., pp. 217, 413). 


6. Next we must consider the aggregation of mesenchyme into a 
superficially extended body, —the formation of a layer. This process 
does not seem to be very common ; one example is seen in Figure 4. 

ec. As the last of these processes of aggregation we have the case of 
aggregation into a mass. This wide-spread ontogenetic process may be 
illustrated by the formation of gemmules in a marine sponge (Figure 5). 
Other examples are found in the formation of the adductor muscles of 


¢ Fig. 3. Cross section of a rat embryo in the upper thoracic region, showing the 
development of the sympathetic nerve (between spinal nerve and carotid artery). 
From A. M. Paterson, 91, Trans. Roy. Soe. Lond., Pl. XXII. Fig. 4. 

Fig. 4. Later stage of the embryo shown in Figure 1. The migrating proto- 
plasm has aggregated itself into a layer at the surface of the embryo. See K. & H., 
Fig. 449. 

Fig. 5. Section of a marine sponge (Esperella), showing a gemmule, a mass of 
aggregated mesenchyme which is about to produce a gemmule (1’), and migrating, 
not yet aggregated mesenchyme (1). After H. V. Wilson (’94, Jour. of Morph., IX. 
Pl XIV.). 


178 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


Lamellibranchs, the muscles of the foot of Gastropods (K. & H., Fig. 
556, s.m.), and the lymph glands and spleen of Vertebrates (M., p. 414). 

The reverse process to the aggregation of mesenchymatous cells is 
their Dispersal, and this has probably been brought about by the 
opposite cause to that producing aggregation. Since, however, this is 
a process taking place in a protoplasmic mass, its consideration must 
be deferred. (See page 194.) : 

We have been considering the different forms into which mesenchy- 
matous elements aggregate themselves in the formation of one body ; it 
now remains to consider the processes taking place between mesenchyme 
and other protoplasmic bodies. Of these processes I recognize at present 
four, viz.: the attachment of mesenchymatous cells to a body, following 
their migration thither ; the encapsuling and interpenetration by a mass 
of mesenchyme ; transportation by mesenchyme; and absorption by 
mesenchyme. 


Fie. 6. Fig. 7. 


4. Attachment of Mesenchyme to another body. This process occurs 
in the union of the muscles of Lamellibranchiata, Annelida, Crustacea, 
and Bryozoa (Figure 6) to the hard parts of these animals, and of ten- 
don to bone, in Vertebrates. 

5. Investment and Interpenetration, by Mesenchyme, of a mass — either 
some other organ of the body or a foreign substance, like a parasite — is 
a not uncommon process. Especially marked is this process in the 
Tunicata (Figure 7), where migrating follicle cells encapsule and finally 


Fig. 6. Sections through the body wall of the Bryozoan, Paludicella; (a) young, 
() adult; illustrating the process of attachment of mesenchymatous muscles to the 
cuticula. 

Fig. 7. Section of the germ disk of Pyrosoma, showing migrating follicular cells 
surrounding the blastomeres. (See K. & H., Fig. 771.) 


DAVENPORT: PROCESSES CONCERNED IN ONTOGENY. 179 


penetrate between the blastomeres, so that it is difficult to tell which 
part of the embryo has been derived from the egg and which part by 
immigration. Compare the 
origin of the cutis in Echino- 
derms (K. & H., Fig. 195), in 
Mollusca (K. & H., Fig. 686), 
and in Vertebrates (M., Fig. 
306), and of the intestinal and 
vascular musculature of Verte- (* Stolon bee 
brates (H.-M., Fig. 185). L ventral side. 
All of these processes have 
this in common, that mesen- 


chyme migrates to an organ 
— vessel, layer, or mass — and 
applies itself closely to it, 
sometimes even penetrating Hrdeade, in 
into the substance of the venaie 
organ. : 

6. Transportation by Mes- 
enchyme has hitherto been 
observed in but few cases. 
The most remarkable instance 
of this process is found in the 
Doliolide, where the buds pro- 
duced from a stolon are trans- 
ported over half the length 


, ; > 3g 80 
and half the circumference of ~ i 35; 
@, a> 
- a OD 
the body by means of mesen 2 SD Satsper tra 
chyme cells, and are finally Re 
deposited, in very regular or- G J XD 
der, on the appendage of the p 
budding individual. (Figures ae sel aiele, 
8 and 9.) By similar means 
g y. By means, Rae a, 


apparently, one end of the 

funiculus of the Bryozoan Cristatella is transported from the dorsal to 
the ventral surface of the corm, as I have attempted to show elsewhere. 
(Bull. Mus. Comp. Zool., XX. 142.) 


Fig. 8. Dorsal view of the posterior part of a large Doliolum “nurse.” Shows 
the buds being transported from the ventral to the dorsal (and posterior) stolon. 
(See K. & H., Fig. 830.) 


180 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


7. Absorption by Mesenchyme. Only of late years have we come 
fully to appreciate the great réle played in ontogeny by the devour- 
ing capacity of mesenchyme. It is now 
fully established that such migratory pro- 
toplasmic bodies — phagocytes — are the 
most important agent in the degenerative 
processes which larve undergo in their 
metamorphoses. 

This is well shown in Insects (Figure 
10), in Bryozoa probably, in Ascidians, and in the frog. 

The secreting activity of mesenchyme has already been classed under 
general processes. No doubt mesenchymatous cells perform various 


Fie. 9. 


pees mp Py Oy : 
MEE Nad: 7 
», L J . 


~ 


Fie. 10. 


other functions in the body besides transportation, digestion, and secre- 
tion, but these either have little effect on the form or concern only 
histogenesis. 


II. ONTOGENETIC PROCESSES OCCURRING IN ELONGATED PROTOPLASMIC 
Bopies — Fisres, THREADS, Corps, TUBULES. 


Falling under this head we may recognize, first, certain general 
changes due to growth, such as increase in length or in thickness. 
These may affect either the whole body or its parts, and may lead 
to a diminution or increase in size. 


Fig. 9. Section through the transported bud of Dolchinia, showing the ameeboid 
transporting cell. (See K. & H., Fig. 859.) 

Fig. 10. Sections through the abdominal imaginal disks of the hypodermis of 
Musea. <A, from the larva. 8B, from the young pupa. In B the phagocytes (p.) 
are in the act of devouring the larval hypodermis (/.), which is replaced by the 
superficial growth of the imaginal disk (d.). K. & H., Fig. 580. 


DAVENPORT: PROCESSES CONCERNED IN ONTOGENY. 181 


We may recognize, in the second place, certain processes which con- 
cern the direction which the elongated body takes and its relation to 
other bodies of the same or of a different kind. This latter group is 
the only one which requires further analysis and illustration. Vacuo- 
lization of cords, such as occurs in blood-vessels, will be considered in 
Section IV. 

In this section we may consider four processes: (1) tropic processes, 
including the turning of elongated bodies towards or from any object ; 
(2) the splitting of such bodies; (3) their anastomosis ; and finally 
(4) their fusion with other organs. 

1. Turning of Elongated Protoplasmic Bodies towards or from an Olyect. 
This is a process which has long been recognized by botanists as occur- 
ring in roots and stems, and as being a response to a stimulus coming 
from outside the organism. Also among hydroids the position of stolons 
and hydranths is often clearly determined by external stimuli. Of organs 
inside of the body, the determination of the direction of growth of nerves 
has been referred by Herbst to the action of a stimulus supplied by the 
organ towards which the nerve grows. Other examples of this process 
are not rare. I will cite a few. Herbst has already referred to the 
case in Turbellaria where unicellular glands of mesenchymatous origin 
send out long processes (the necks), which gain their appropriate con- 
nections with the other organs. So, too, the shell-gland of Crustacea, 
which arises from an aggregation of mesenchyme (K. & H., p. 377) 
secondarily sends out a stalk which makes connection at the appropriate 
place. The principal blood-vessels of Vertebrates arise in the area vascu- 
losa and subsequently grow into the embryo, following certain prescribed 
paths (M., pp. 215, 216). From these vessels others in turn bud out, 
progressing towards their destined organs. According to Field (791, 
Bull. Mus. Comp. Zo6l., X XI. 222), the pronephric duct of Amphibia, 
arising in mesoderm, grows at its posterior end, secondarily fusing with 
the cloaca. Again, the tubules of the metanephros, according to some 
authors, bud out of the blind end of the ureter, and in their further 
growth hit exactly the independently formed Malpighian capsules ly- 
ing in the mesenchyme. In all these cases we have the elongating 
body clearly turning towards the object with which it is destined to 
unite. 

Even under some abnormal conditions we have this process taking 
place ; for instance, when a parasite lodges in any organ of a Vertebrate, 
new vessels are formed, which grow out towards the source of irritation. 
(Cf. Roux, ’81, Der Kampf der Theile, pp. 150, 151). 


182 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


2. Splitting of an Elongated Protoplasmic Body (cord or tubule). We 
may recognize here two sub-processes: (a) splitting at the tip, and 
(6) splitting along the whole length. 

a. As is well known, nerves grow out as blunt stalks which repeatedly 
divide at their ends. Paterson has shown that some of the spinal nerves 
split at the somato-splanchnic angle, as shown in Figure 11. His (’88, 


Arch. f. Anat., Jahrg. 1887, p. 376) shows in many cases that the split- 
ting takes place where the end of the growing nerve strikes a rigid organ. 
Thus, he says, when the third branch of the trigeminus strikes Meckel’s 
cartilage, it divides into the ramus lingualis and the ramus mandibularis ; 
and when the hypoglossal meets the jugular vein, it divides into its de- 
scending and lingual branches. The way in which the rigid organs act 
to produce splitting is thus explained by His. Pre-existing resistant ob- 


” 


jects may be considered as the “ Motivs” of division; for as a nerve 


Fig. 11. Cross section of a mouse embryo in the lumbar region, showing the 
splitting of the spinal nerve at the upper angle of the celom. After Paterson, 791, 
Trans. Roy. Soc. London, CLXXXL, Fig. 6. 


DAVENPORT: PROCESSES CONCERNED IN ONTOGENY. 183 


stem strikes a cartilage or a vessel, its fibres are bent in different direc- 
tions and the stem divides. The resisting objects are thus considered 
by His to act in a mechanical, i.e. direct way. The results are, however, 
equally explicable by the response-to-stimulus theory. 

The splitting which occurs at the blind ends of developing blood-ves- 
sels and excretory tubules, and the repeated divisions of many glands — 
salivary gland (M. Fig. 334), liver, and lungs (M., Fig. 445) — are exam- 
ples of this process. Other cases are found among Invertebrates, as, for 
example, the tentacles of many Cnidaria (K. & H., Figs. 27, 31), “roots” 
of Sacculina, and liver branches of Limulus (K. & H., Fig. 338). 


SS 


—mSN 
<i | 


b. The second case, that of division of a tubule thronghout its 
entire length, is illustrated in the development 
of the segmental duct in some Vertebrates, and 
in the separation of aorta and pulmonary artery. 
(Figure 12). 

3. Anastomosing, or the process of fusion of 
similar threads, thus forming a network, is of 
wide-spread occurrence. It is exemplified in the 
development of nerves (Figure 13) and blood-ves- 
sels in Vertebrates, and in some glands, especially 
the vertebrate liver. 

4. Fusion with other Organs. The process of 
fusion of diverse organs will be studied in greater 
detail in other sections of this paper. It occurs, 
for instance, at the close of the process of growth 
of the neck of a mesenchymatous gland (or other independently arising 
tubule) towards its insertion. 


Fre. 18. 


Fig. 12. Sections at different levels through the cardial aorta of a human 
embryo of 11.5 mm. The lowest section is at the left: the highest at the right; 
the separation progresses from above downwards. a, aorta; p, pulmonary artery. 
See M., Fig. 293. 

Fig. 13. The cervical and three of the thoracic spinal nerves of a human embryo, 
showing the origin of the thoracic plexus. See M., Fig. 560. 


184 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


III. OntToGENETIC PROCESSES OCCURRING IN PRoToOPLASMIC LAYERS. 


The ontogenetic processes occurring in protoplasmic layers may con- 
cern one layer only, or two or more layers acting in relation to each 
other. 

The processes which take place in one layer are principally growth 
processes affecting on the one hand the area, on the other the thickness 
of the layer. 

III*%. Of the growth processes affecting area, we may distinguish 
(a) such as take place in the walls of a hollow sphere or cylinder from 
(8) such as take place on a plane or warped surface. 

a. The areal growth occurring in a hollow sphere or cylinder (sac) 
may be equal in all the axes or elements of the wall, leading merely to a 
change of size of the sac; or it may be unequal in the various axes or 
elements, producing a change in form. It is 
this latter group which especially interests 
us, and it will therefore be further analyzed. 

1. We may recognize three cases in the 
differential growth processes occurring in 
a sac: (a) unequal growth in the different 
axes ; (6) unequal growth at the poles of the 
axis; (¢) unequal growth in the various me- 
ridians of the sac. 

These three processes deserve illustration. 

a. By the process of excessive growth in a 
certain axis we have the ellipsoidal form pro- 
duced from the spherical ; as in the Sycandra 
larva (Figure 14)+ in planule (K. & H., Figs. 
14, 30, 32, 51); in the larve of various worms 
(K. & H., Figs. 144, 158, 159); in the Echinoid 
blastula (K. & H., Figs. 173, 176); and in the 
larvae of some Mollusca (Figs. 542, 576, 593, 
596) and Bryozoa (Fig. 702). 

6. Through unequal growth at the two poles of (e. g. the chief) axis, 
we have produced such ovoidal forms as planule, in which not only is 
the chief axis elongate, but one pole has grown more than the other. 
(Figure 15.) By unequal growth at the poles are produced also such 


Fie. 14. 


Fig. 14. Sycandra embryos. A, 8-cell stage; B, larva. See K. & H., Figs. 2 
and 3. 


DAVENPORT: PROCESSES CONCERNED IN ONTOGENY. 185 


club-shaped pouches as the “club-shaped gland” of the Amphioxus 
larva (K. & H., Fig. 875). 

e. Through unequal growth of the different meridians of a spheroidal 
or cylindrical wall we have produced from the spheroid an apparent 
flattening on one face, such as occurs, for example, in various cases of 
“epibolic gastrulation.” In Figure 16 this process is illustrated in the 
spheroidal egg of a Heteropod. (Cf. K. & H., Figs. 66, 541, 738.) 


By an excess of growth along the meridians of one side of a cylinder 
we have produced such apparent rotations of the internal organs us 
occur in Crinoids and Endoprocta. (K. & H., Figs. 218, 732.) 

8. Let us now consider the unequal areal growth of a part of a 
bounding layer taken so small that it may be considered as nearly a 
plane or a warped surface.’ We shall find that the processes taking 
place in such a part are among the commonest and most important 
of all occurring in ontogeny. 

This unequal growth may result, either (2) in parts formerly lying in 
one plane moving into different planes; or (3) in parts formerly lying 
in one line moving out of that line. 

2. It has long been recognized that, when in a layer excessive growth 
takes place over a restricted area, the result is that the excessively 
enlarged area can no longer occupy its former territory. Since its 
periphery remains relatively constant while the included layer increases 


Fig. 15. Section through the planula of Zquorea, to show the greater expanse 
at one pole than at the other. See K. & H., Fig. 14. 

Fig. 16. Sections through young embryos of Firoloides, showing how the condi- 
tion in stage B has been derived from that of stage A by greater growth along the 
meridians on the left than on the right. See K. & H., Fig. 584. 


186 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


in area, the latter is compelled to fold, producing, according to the point 
of reference, an elevation above or a depression below the general level. 


eS 
Fre. 17. 


So important relatively is the process of folding that it has apparently 
been regarded by some authors as almost the sole developmental pro- 
cess. How important a r6le it 
plays is well illustrated in Figure 
18, where (excepting the nerve 
masses and the heart, which are 
unrepresented) all of the differ- 
entiated organs of the squid 
which at this stage lie in the 
sagittal plane are represented. 
And these are seen to have 


: arisen by the foldings of the 
Radute-F a 

Sac. 2 a 
Sass y AY 
Saige 


unilaminar blastoderm which 
covers the yolk. The connec- 
tions of the otocyst and the 
shell-gland with the exterior 
are now lost, but fin, mantle 
margins, funnel, and the various 
Fic. 18. appendages of the alimentary 

tract, as well as the tract itself, 

are all seen to be arising as foldings of the blastoderm. This view does 


Fig. 17. Diagram representing the process of folding as it occurs in proto- 
plasmic layers. By excessive growth between a and 2}, the fold aob is pro- 
duced. 

Fig. 18. Sagittal section of an embryo of Loligo vulgaris, showing the origin of 
organs through foldings of the blastoderm. From K. & H., Fig. 673. 


DAVENPORT: PROCESSES CONCERNED IN ONTOGENY. 187 


not include the lateral organs, —eye vesicles, gills, and arms, — which 
arise in a similar fashion as folds. 

Based upon the form of the growing area, two classes of folds may be 
recognized : — 

(a). Pockets, from a circular area. 

(4). Linear folds, from an elongated area. 


Tite TMX 
[ae —— ee ih 


Good examples of the formation of pocket folds are seen in many cases 
of gastrulation, and in the otocyst, shell gland, salivary gland, and sepia 
sac of Figure 18. 


Fig. 19. Two stages in the development of the epidermis and neural tube of the 
chick. Both magnified to the same extent. (From W. His, ’94, Arch. f. Anatomie, 
Jahrg. 1894, pp. 74, 76 ) 

VOL. XXVII. — NO. 6. 9 


188 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


Typical linear folds occur in the formation of the neural tube of most 
Vertebrates, of the lateral line of fishes, and of the atrium of Amphioxus. 
(K. & H., Fig. 881.) 

3. Inequalities of growth in different parts of the layer may take 
place, so that parts originally lying in a line move out of that line. By 
this means are produced folds lying in the plane of the layer, — folds com- 
parable to those made by an advancing wave front on entering a bay. Such 
folds may occur in the bounding line of the growing layer, as happens, it 
is alleged by some, in the overgrowth of the yolk by the blastoderm, — 
a process by which the crescentic notch is produced on the edge of the 
blastoderm, Again, such folds may occur in the midst of a layer, pro- 
ducing a relative displacement of the points lying in that layer. To this 
process may be referred the remarkable rotation of tbe gill slits and 
mouth of Amphioxus, —the gill slits rotating from the right to the 
left side of the larva, the mouth from the left side towards the mid- 
ventral line. 

II1. The processes affecting thickness of the layer are of very com- 
mon occurrence, and are of two kinds, — (4) processes leading to an 
increase, and (5) those leading to a diminution in thickness. These 
variations in thickness may be general, extending over a considerable 
part or the whole of the area under consideration, or they may be 
restricted to a small part of that area. 


Nerve cord. 
‘ 


4, The process of thickening is illustrated in the lateral walls of the 
neural tube of Vertebrates (Figure 19), and, localized, in the formation 
of the neural tube in Teleosts (Figure 20). Other examples are seen in 


Fig. 20. Cross section of the blastoderm of a bony fish (Serranus atrarius) to 
show the linear thickening of the ectoderm to produce the nerve cord. (From H. 
V. Wilson, 791, Bull. U. S. Fish Commission, IX. Plate XCV.) 


asi a ee ooo 


DAVENPORT: PROCESSES CONCERNED IN ONTOGENY. 189 


the papillz which precede the formation of feathers, and in the ingrowing 
follicles which form hairs. 

5. A general thinning of the walls of the organs is found to accompany 
the development of many Invertebrates. Figure 21 illustrates this fact. 


IM gels WAL 


In Figure 19 a general thinning of the epidermis is seen in passing from 
Stage a to stage B. A good example of local thinning is seen in the 
formation of the ependyma of the roof of the brain and medulla. 

The processes of thickening and thinning may go on side by side, as in 
the formation of the lens of the vertebrate eye (Figure 22), where the 
outer and inner layers of the lens are at an early stage of nearly equal 
thickness, but where, in the later Stages, the outer layer becomes thin, 
and the inner layer enormously thick. 

IlI*. Next, we must consider the processes by which a single con- 
tinuous layer experiences an interruption in its continuity. This may 
take place (6) by a complete atrophy of a part of the membrane, thus 
forming a hole or rift in it, or (7) by a separating off of a certain piece 
from the membrane. 


Fig. 21. A, younger, and B, older Trochopore stages of the annelid Eupomatus 


- uncinatus, seen in sagittal section. Drawn to same scale. Outlines copied from 


Hatschek, ’86, Arb. Zool. Inst. Wien, VI. See also K. & H., Fig. 118. 


190 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


6. The former process occurs typically in the ontogeny of reptiles, and 
perhaps of mammals, where the middle of the layer of “ primary ento- 
derm ” roofing the yolk cavity atrophies, making a great hole in the layer. 


Fig. 22: 


7. The latter process is represented in Hatschek’s familiar figure of 
the formation of the neural tube of Amphioxus (Figure 23), where the 


ree 23: 


medullary plate is shown as falling 
below the general level without the 
intervention of a fold. Compare the 
formation of the ‘body plate” of 
the Nemertine Lineus, K. & H., Fig. 
105, and gastrulation in Apis, K. & 
H., Fig. 495. 

III*. Finally, we must consider the 
important processes involving two or 
more protoplasmic layers. Such layers 
may move either towards or from each 
other. We will here consider only the 
former group of cases. 


8. The mutual approach and fusion of layers may be called con- 


Fig. 22. A. Section of eye of chick embryo on third day. (See M., Fig. 413.) 
B. Section of eye of rabbit embryo, 13 days old. (See M., Fig. 409.) 7. l. 1, inner 
lens layer; o././., outer lens layer; r. c. retinal cup. 

Fig. 25. Section across an Amphioxus embryo, showing the medullary plate, 
m. p., which has sunk below the level of the ectoderm, producing a discontinuity in 


the latter. From H.-M., Fig. 69. 


DAVENPORT: PROCESSES CONCERNED IN ONTOGENY. 191 


erescence. We may distinguish three sub-processes under this head, 


® 
® 
Entod. ae’ ee 2 
° 0® ee } » e 
8 e% e ~ A e@ 22.0 
&%. PO e e eg AES 
eo ® Se @5q0° e 
O) @ ee 79 
Co & Cy Q 
Leche. 11} 08a toes? > 
a fe ar 
Z r g 
Fig 24. 


depending upon whether (a) the concrescence takes place along the 
free margins of layers, or (6) along their sur- 


faces, or, finally, (c) along the edge of folds. LR pe 
This concrescence is usually quickly fol- Ai eae 
lowed by other processes which we will 


consider later. 

a. The concrescence of layers by their 
free edges is illustrated in the cases of the () 
growing together of the free edges, x and B. 
x’, of the ectoderm in the Amphioxus em- 


bryo at a stage a little later than that 

shown in Figure 23. (K. & H., Fig. 504.) jf 
b. The concrescence of layers flatwise is 

illustrated in the formation of the verte- oO Cc. 

brate mouth when the anterior end of the 


entodermal sac comes in contact with the 
ectoderm. Likewise in the formation of the 


gill slits of Vertebrates the broad bottoms Ss 
of the entodermal sacs move to the ecto- 
derm. (Figure 24, I, II.) 2. 


c. Concrescence along the edges of two 
folds is perhaps the commonest of these 
three forms of concrescence. It is that by 
which in Vertebrates the neural tube is 


Fie. 25. 
closed (Figure 25, A, Bb, C); ectodermal 


Fig. 24. Part of a frontal section through an embryo of Acanthias vulgaris, of ° 


about the stage of Balfour’s Stage I. Shows 3 stages in the formation of the gill 
slit, I, II, illustrating concrescence of layers flatwise; III, perforation. Original. 
From a preparation kindly lent me by Mr. H. V. Neal. 

Fig. 25. Cross sections through the neural tube of embryo frogs of different 
ages, showing the concrescence of the lips of the medullary groove (A, B, C), and 
(D) the final separation of the upper and lower layers of the fold. After H. H. 
Field, 91, Bull. Mus. Comp. Zool., X XI. No. 5. 


qe 


. 


192 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


pockets, like the lens and otocysts, become transformed into closed sacs ; 
and grooves, like those of the lateral line, become transformed into canals. 
In Invertebrates also this process is a very common one, being exempli- 
fied in the closure of the blastopore, in the closure of the amniotic cavity 
in Insects (K. & H., Figs. 474, 475, 484), and in the formation of optic 
and otic vesicles generally (K. & H., Figs. 377, 630, 681-683). 

The end of the process of concrescence proper is a fusion of the two 
concrescing layers, whether the concrescence is occurring along free 
edges, flatwise, or along the edges of folds. (Fig. 25, C, Fig. 24, IL.) 

9. Frequently this process is followed by another one; viz. the per- 
foration of the fused layers (Fig. 24, III), or the separation of the 
upper and lower components of the folds when two folds have been 
conerescing (Fig. 25, D). By means of perforation the two spaces 
separated by the fused walls are put into communication with one 
another. By means of separation a pocket becomes a closed sac, and 
a groove becomes a tube. 


IV. ONTOGENETIC PROCESSES OCCURRING IN PROTOPLASMIC MASSES. 


These may be classed into three categories according as the most 
prominent change produced is (IV*) in volume, (IV”) in form, or 
(1V°) in number of masses. 

IV*. 1. Under the first group are included changes produced by 
growth which is not uniform in all parts. Thus the growth may be 

prevailingly along one axis, by which 

Spinal cord. means a cylindrical mass is derived 

: from a spherical one (embryos of Dy- 
ciemide, K. & H., Fig. 99), or it may 
be excessive at one pole (gemmules of 
sponges, H. V. Wilson, ’94, Jour. of 
Morphol., LX., Pl. XVI.), or along one 
meridian. Again the growth may be 
more localized, being confined to a 
\ small area or to a line; as, for in- 
Fic. 26. ; stance, in the case of the mesodermal 

core of the appendages of Arthropods 

(K. & H., Fig. 371), and of Vertebrates (Figure 26, “limb-bud”). By 


Fig. 26. Cross section through embryo of a Teleost, Fundulus, showing origin 
of the pectoral limb-bud as a solid outgrowth of the somatopleure. After E. R. 
Boyer, 92, Bull. Mus. Comp. Zool., XXIII. No. 2, Fig. 58. 


DAVENPORT: PROCESSES CONCERNED IN ONTOGENY. 193 


this process of localized solid growths the principal differentiations of 
Phanerogams occur. 

IV’. 2. An important change of form 
of a protoplasmic mass may occur inde- 
pendently of growth by a rearrangement of 
the nuclei of the mass. 

Good examples of this process are found 
in the development of the larva of Lucer- 
naria (Figure 27); in the development of 
Ctenophores (K. & H., Fig. 67); and in 
the changes of form occurring in the 
“ectodermal basal plate” of Salpa (an 
apparent syncytium), according to the 
figures of Heider (’95, Abh. Sencken- 
berg. naturf. Ges., Bd. XVIII. Figs. 32, 
40, 41). In so far as this process involves the migration of nuclei, it 
is clearly closely related to Process I. 1. 


Fie. 28. 


Fig. 27. A, B, C, are three successive ontogenetic stages of Lucernaria. The 
transformation from stage 6 (36 nuclei in the section) to stage C (33 nuclei in 
the section) is due to a rearrangement of the nuclei. See K. & H., Fig. 49. 

Fig. 28. 2, dispersal of elements of the gemmule A, which was formed, as 
illustrated in Figure 5, by the aggregation of similar mesenchymatous elements. 
This process precedes the development of the gemmule into a larva, and accom- 
panies the imbibition of water by the gemmules. After H. V. Wilson, ’94, Jour. 
of Morphol., [X., Plate XVI. 


194 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


3. Another way in which the mass changes form without growth is 
by vacuolization. Through vacuolization there arise, e. g., the blastula in 
many eggs, the secondary body cavity in many Invertebrates (K. & H., 
Fig. 131, 689), and in Bryozoa the cavities of the bud. This process of 
vacuolization, which I introduce for convenience at this place, is not 
confined to masses. Many cords by vacuolization (canalization) become 
tubules, and many layers become divided into two. Of vacuolated cords, 
I need refer only to the formation of capillaries in Vertebrates and of 
nephridia in Invertebrates; of vacuolated layers, to the origin of the 
celom in most Vertebrates. In all cases, the acquisition of a mass 
of water at the centre causes a rearrangement of the nuclei. 

4, Perhaps this is the most fitting place to mention the process of 
Dispersion of elements, which occurs not only in bodies originally 
formed by aggregations of mesenchymatous elements (Fig. 28), but 
also in /ayers having an epithelial origin, e. g. the ectoderm of Dis- 
tomum. (K. & H., Fig. 88.) 

IV*. The remaining processes occurring in protoplasmic masses are of 
such a nature as to alter the number 
of masses. We can distinguish, corre- 
spondingly, two classes: the first in- 
cluding such as have to do with the 
formation of two masses from one, 
through division ; the second including, 
those which have to do with the union 
of two masses into one. The first, 
then, are division processes; the sec- 
ond, fusion processes. 

5. Under the first head we may in- 
clude the process of constriction, by 
which two more or less independent 
masses arise from one. An _ illustra- 
tion of this is found in the case 
of embryonic fission described for 
some Bryozoa by Harmer (Figure 29). 
Closely allied to this is the process of 
sloughing off of a part of the body in 
metamorphosis, one of the most striking instances of which is shown in 


Fig. 29. Section through a brood chamber of Crisea eburnea, showing the con- 
stricting off of secondary embryos from the primary embryo. After Harmer, 93, 
Quart. Jour. Mic. Sci., XXXIV., Plate XXIII. Fig. 11. 


DAVENPORT: PROCESSES CONCERNED IN ONTOGENY. 195 


Sacculina, where the thorax and abdomen are thrown off, the head alone 
persisting to complete the metamorphosis. 


6. Another division process is that of splitting of the mass. This is 
illustrated by the case of the optic mass 
of the lobster (Figure 30), which splits 
into an outer and an inner part. Com- 
pare the origin of the nervous system of 
Peripatus, K. & H., Fig. 442, B. 

7. Under the second head, fusion of 
contiguous masses, we may place such 
cases as that of the union of indepen- 


dently arisen ganglionic masses, such as y, 

7 a ‘ ; Ventral 
Morgan describes for the Pantopod, Pal- Bangla. 
lene (Figure 31). Fie. 31. 


Fig. 30. Sections through three stages in the development of the compound 
eye of the lobster after G. H. Parker. In 6 and C the mass is seen to be splitting 
into the retina and optic ganglion. See K. & H., Fig. 263. 

Fig. 81. Ventral part of sections across Pallene embryos. A, earlier stage, 
showing the paired neural invaginations ; 5, later stage, ganglia fused. See K. & H., 
Figs. 409, 410. 


196 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


RECAPITULATION AND GENERAL REMARKS. 


We may now, in recapitulation, arrange in tabular form the differen- 
tial processes which we have recognized. 
I. Processes occurring in mesenchyme. 
1. Migration of nodal thickenings, p. 175. 
2. Free migration of amoeboid bodies, p. 176, 
a. from a layer. 
6. through fluid-filled spaces. 
3. Aggregation of mesenchyme, p. 176. 
a. into a thread. 
b. into a layer. 
c. into a mass. 
4. Attachment of mesenchyme, p. 178. 
5. Investment and interpenetration, p. 178. 
6. Transportation, p. 179. 
. Absorption, p. 180. 
II. Processes occurring in protoplasmic threads or tubules. 
1. Tropism, p. 181. 
2. Splitting, p. 182. 
3. Anastomosing, p. 183. 
4. Union with other organs, p. 183. 
III. Processes occurring in protoplasmic layers. 
III*. Processes affecting area. 
a. Processes occurring in the wall of a sac. 
1. Excessive growth of particular parts, p. 184. 
a. along one axis. 
b. at one pole. 
e. along one meridian. 
B. Processes occurring in a plane or warped surface. 
2. Formation of perpendicular folds, p. 185. 
a. Pocket folds. 
b. Linear folds. 
3. Formation of folds in the plane of the area, p. 188. 
ITI®. Processes affecting thickness. 
4, Thickening, — general or local, p. 188. 
5. Thinning, — general or local, p. 189. 
III°. Processes affecting continuity. 
6. Atrophy, p. 190. 
7. Detachment of a piece from a layer, p. 190. 


=~] 


DAVENPORT: PROCESSES CONCERNED IN ONTOGENY. LF 


III*. Processes affecting two or more layers. 
8. Concrescence, p. 191. 
a. of free edges. 
b. of surfaces. 
ce. of edges of folds. 
9. Perforation, p. 192. 
IV. Processes occurring in protoplasmic masses. 
IV*. Effecting especially change of volwme, 
1. Excessive growth of particular parts, p. 192. 
b. general. 
e. local. 
IV. Effecting especially change of form. 
2. Rearrangement of nuclei, p. 193. 
3. Vacuolization, p. 194. 
4. Dispersion of elements, p. 194. 
IV°. Effecting especially change in number. 
. Constriction, p. 194. 
. Splitting, p. 195. 
. Fusion,.p. 195. 


I oS Or 


The processes here ennumerated may be for the most part grouped 

under three general heads : — 
I. Taxice processes. 
II. Tropic processes. 

III. General growth processes. 

Under Yaaxic Processes I include such as are accompanied by free 
migration of protoplasmic bodies, or by the flowing of protoplasm from 
one part of the whole body to another part. 

Under Tropie Processes I include such differential ani processes 
as result in a turning of protoplasmic bodies (threads or folds) towards 
one another or towards another protoplasmic body. 

By General Growth Processes 1 mean those differential growth pro- 
cesses which are not included under tropic processes. 

To the category ‘‘taxie processes” may be provisionally assigned 
Nos. I. 1, 2, 3, 4, 5, and 6; III. 4 (in part) and 5 (in part), 7 (4), 9 
mye 2, 4, 5,6, 7. 

To the category “tropic processes” may be assigned Nos. II. 1, 2, 3, 
oe LIT. 8 9. 

To the category “ general growth processes ” may be assigned Nos. III. 
I, 2, 3, 4 (in part) and 5 (in part), 6; IV. 1 and 3. 


198 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


It will be noted that No. I. 7 (absorption by mesenchyme) is not 
assigned to any one of the three categories ; and that certain other pro- 
cesses (III. 7, Detachment from a layer; II. 2, Splitting; III. 4 and 5, 
thickening and thinning of a layer; and IV. 3, Vacuolization) are of so 
donbtful a nature that, although assigned to the special categories, this 
assignment can be regarded as provisional only. 

The process of absorption does not readily fall into one of the three 
categories, and at the same time it does not seem worth while to erect a 
special category for it. 

As for the doubtful cases, the doubt is not whether they are referable 
to one of these categories, but rather in knowing in which one to place 
them. 

Regarding the three general categories, it has long been recognized 
that taxic and tropic processes are responses to stimuli. It has not 
been so generally recognized that all growth processes are such. A 
moment’s consideration will, however, make this probable. 

Let us consider for a moment what it is that controls differential 
growth, — What makes one part of a membrane grow faster than another, 
causing a folding of that part ? 

Inequality of growth is clearly not due to inequality of food supplied, 
since folds arise in uniformly nourished membranes, — bathed, that is to 
say, uniformly by the nutritive fluids. Jt must therefore be due to ine- 
quality of the activities which lead to growth; namely, the taking in of 
food and its assimilation, and the imbibition of water. Now it is our fun- 
damental assumption that activities of all sorts, including ingestion and 
imbibition, are responses to stimuli. In so far, then, as differential 
growth is dependent upon the inequality of these activities in diifer- 
ent parts of the membrane, it is dependent upon stimuli acting upon 
that membrane. 

Whenever the activities are diverse in the different parts of a mem- 
brane, it must be either that the stimulus applied to the different parts 
is diverse, or, if not, that the protoplasm is diverse in its different parts, 
for what the result shall be depends upon two factors, — the quality of 
the stimulus and that of the protoplasm. 

Let us now consider somewhat more in detail the taxic and tropic 
processes. As is well known, the stimuli which control these movements 
result either in migration towards the source of the stimulus or away 
from it, so that positive or negative taxis or tropism occurs. In ontogeny 
it is often impossible to say where the source of stimulation is, and 
therefore whether the tactic or tropic movements are +- or —. Cer- 


DAVENPORT: PROCESSES CONCERNED IN ONTOGENY. 199 


tain criteria may, however, be employed in some cases to determine 
this. Thus, where many migratory bodies move towards a common 
point, or where a thread or tubule makes its way to a distant point, 
we may believe that a positively tropic stimulus is exercised by that 
point. Also, where two similar parts move towards each other, it is 
probable that a + stimulus is exerted by both; where, on the other 
hand, they mutually withdraw, it is probable that a mutual negative 
stimulus emanates from both. 

With these criteria in mind we may classify some of the taxic and 
tropic processes as + or —, and this I have attempted to do in the 
following table: — 


PROBABLE RESPONSES TO POSITIVE] PROBABLE RESPONSES TO NEGATIVE 


STIMULI. STIMULI. 
Taxic. Tazic. 
I. 3. Aggregation of mesenchyme. I. 2,a. Migration from a layer. 
I. 4. Attachment of mesenchyme. UI.4. Thinning of a layer (when due 
I. 5. Investment and interpenetra- to flowing of the protoplasm 
tion. from a point). 
I. 6. Transportation. III. 9. Perforation. 
III. 4. Thickening of a layer (when|IV.4. Dispersal of elements. 
due to flowing towards one | IV. 5, 6. Separation of masses. 


point). 
IV. 7. Fusion of masses. 


Tropic. 
II. 1. Turning of thread. 
II. 5. Anastomosing. 
IL. 4. Union with other organs. 
IIL. 8. Concrescence. 
PROBABLE RespONSES TO EITHER -+- oR — STIMULI. 


I.1. Migration of nodal thickenings. 
I. 2,6. Free migration of ameboid bodies. 
1V.2. Rearrangement of nuclei. 


It is not too much to believe that the foregoing hypothetical interpre- 
tation of the ontogenetic processes lies within the possibility of experi- 
mental test. Just as the control of the migration of amoeboid bodies in 
the adult has been undertaken with success, so may we hope to control 
the tropic and aggregation phenomena of ontogeny. By experiment alone 
can the causes of the developmental processes be determined. 


CAMBRIDGE, Mass., May 1, 1895. 


the Museum of Comparative Zodlogy — 
peer > “. y - i 


AT HARVARD COLLEGE. 
2 Vou. XX VII. © No. T. 


THE EARLY EMBRYOLOGY OF CIONA INTESTINALIS, 
See FLEMMING (L.). ‘ 


« 


. 


_ CAMBRIDGE, MASS., U.S. A.: 
PRINTED FOR THE MUSEUM. 


F Fi 


pe G x t < 


v, 1896 * 


Bulletin of the Museum of Comparative Zoology 
AT HARVARD COLLEGE. 
Wor, XX VII. No. T. 


THE EARLY EMBRYOLOGY OF CIONA INTESTINALIS, 
FLEMMING (L.). 


By W. E. Caste. 


With THIRTEEN PLATES. 


CAMBRIDGE, MASS., U.S. A.: 


PRINTED FOR THE MUSEUM. 
JANUARY, 1896. 


FEB 17 1296 


No. 7.— The Early Embryology of Ciona intestinalis, Flem- 
ming (L.)1 By W. E. Castie. 


CONTENTS. 
PAGE PAGE 
I. Introduction. . . 2 208 A. Early Stages of Gastru- 

II. Material, Life Uitocy pene e205 lations; =e » . 243 
mM (Methods . . . . ait fy alo (a) 112-cell Stace 3s. « 248 
1. Killing, Presceyation 5 al} (6) Differentiation of 
2. Decortication, Staining, the Principal Or- 

Mounting . . . 214 gans as seen at 
IV. Maturation and Fertilization ali? the 112-cell Stage 245 
] Summary on Maturation and a. Topographical. 245 
Fertilization . ... .. 228 B. Histological . 247 
V. Polarity of the Egg . .. . 224 B. Later Stages of Gastru- 
Summary on Polarity of the lation =. = ey aese 
eos. 5 Be (a) From 112-cell to 
VI. Cell Lineage of fie. Sinbiye . 226 128-cell Stage . 249 
eNomenclaturews0).. & %04))226 (b) Closure of the Blas- 
2. Cleavage. . . 227 toporeysn if 252 
A. Early Stages Ff Gleay C. Summary on Gasrata 
pegs ee We . 227 (Mor Ge 5 4a! 
(a) To 24-cell Sue . 227 4, Formation of fie Taeve . 263 
(b) Summary on Early Summary on Formation 
Cleavage Stages. 234 of, the” Larva ae 265 
B. Later Stages of Cleay- VII. Discussion of some Theoreti- 
BOC ee 235 cal Questions . . . 267 
(a) From 4. ea A “46. A. Origin of the Germ Laven 
cell Stage 235 of Chordates. . . . 267 
(>) 48-cell Stage . . . 287 B. The Celom Theory . . 271 
| (c) 64-cell Stage. . . 239 C. Ancestry of the Chordates 272 
t (d) 76-cell Stage . 240} VIII. Conclusions . . 4 aad 
) (e) Summary on Tetet IX. Table of Cell Tae Peo Ze 
Cleavage Stages. 242/ Literature Cited . . . .. . . 276 
3. Gastrulation. . . . . . 243! Explanation of Plates . . . . . 280 


I. INTRODUCTION. 


So long ago as 1866, Kowalevsky wrote, ‘‘ Die Entwicklungsgeschichte 
der Ascidien wurde schon vielfach studirt.” If this statement was true 


1 Contributions from the Zodlogical Laboratory of the Museum of Comparative 
Zodlogy at Harvard College, under the direction of E. L. Mark, No. LII. 
VOL. XXVII. — NO. 7. 1 


204. BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


then, it is doubly so now, for the literature of the subject has since that 
time multiplied many fold. Nevertheless there still remain many un- 
settled questions regarding the embryology of the Tunicates. Concern- 
ing so fundamental a point as the derivation of the primary germ layers 
in the embryo, quite contradictory opinions have been expressed within 
the last ten years by observers of world-wide reputation. 

I undertook the inquiry, the results of which are recorded in the fol- 
lowing pages, in the hope of being able to throw light on this disputed 
question by the study of other forms than those which had been most 
carefully examined, and by the application of new methods to the prob- 
lem. A short experience convinced me that the only method which could 
yield positive conclusions was that of cell lineage, a method which has 
been applied so successfully to the study of annelid and molluscan em- 
bryology by a number of observers, and had already been employed to a 
limited extent in the study of ascidian embryology by Van Beneden et 
Julin (84), Seeliger (’85), and Chabry (’87). 

It soon became clear to me that some of the conflicting statements 
made by my predecessors arose from errors on their part due to incor- 
rect orientation of certain stages. The nature of these errors I have 
fully explained in a preliminary communication (Castle, 94). A further 
study of the embryonic history, cell by cell, through the periods of cleav- 
age and gastrulation, and even down to the differentiation of the several 
larval organs, has led me to conclusions somewhat at variance with those 
of earlier investigators regarding the origin of the primary germ layers 
and the organs derived from them. One of the most important of these 
conclusions is that the mesoderm of Ascidians — and probably also that 
of Amphioxus and the Vertebrates — is derived in part from the primary 
entoderm and in part from the primary ectoderm. The grounds on 
which this conclusion rests are set forth in the later portions of this 
paper; in the earlier part of the paper I have recorded some observa- 
tions on the maturation and fertilization of the ascidian egg. 

It gives me pleasure to acknowledge in this place my very great obli- 
gations to Professor E. L. Mark for direction and kindly criticism of my 
entire work. My best thanks are also due to Dr. Alexander Agassiz, in 
whose laboratory at Newport the material for my studies was chiefly 
collected, and to Colonel Marshall McDonald for numerous courtesies 
extended to me at the United States Fish Commission Station at 
Wood’s Holl. 


OU 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 20 


II. MATERIAL, LIFE HISTORY. 


The material for this study was collected in the months of August and 
September of two successive seasons, 1893 and 1894. The species em- 
ployed seems to be, beyond question, the Ciona intgstinalis of Flemming, 
a classical object of study on the other side of the Atlantic. It was made 
the subject of an extensive monograph by Roule (84) ; its larval history 
has been studied by Kowalevsky (’66 and ’71) and by Willey (93); its 
cleavage stages by Samassa (’94); its fertilization stages by Boveri (’90); 
and the formation of its egg envelopes by Fol (’84). Loeb (91) also 
has employed it in certain physiological investigations. The specimens 
which I collected at Newport answer fully to Roule’s detailed descrip- 
tions of the species. The large size (8-10 cm. long) attained by individ- 
uals at Newport under favorable conditions confirms Roule’s conjecture 
that the forms described from the United States as Ascidia ocellata by 
Louis Agassiz, as A. tenella by Stimpson (’52), and as Ciona tenella by 
Verrill (71) were only small-sized individuals of Ciona intestinalis. 

Specimens were obtained by me from two different localities just 
within the entrance of Narragansett Bay. The animals were usually 
found adhering to the under side of stones at a depth of from a few 
inches to a few feet below low-water mark. Upon removal to the labora- 
tory they were carefully washed and placed in aquaria whose water was 
kept fresh by a jet of air. Once a day the water was changed, and the 
aquaria thoroughly cleaned, to prevent the accumulation of bacteria or 
other possibly injurious organisms. ‘This painstaking treatment was 
probably unnecessary, for the animals are very hardy and bear ill-treat- 
ment well. For example, I have kept specimens for weeks at a time in 
small glass aquaria without change of water, and the only signs of mis- 
use which they exhibited were a slight shrinkage in size and a greatly 
diminished production of eggs, — both symptoms referable to an insuffi- 
cient food supply. 

Ciona, like all other Tunicates, is hermaphroditic, and the number of 
eggs produced by a single adult individual in the course of a season must 
be enormous. Often hundreds are deposited in a single night. Under 
normal conditions each adult individual, during the summer months, 
lays eggs once in every twenty-four hours, with the regularity of the 
sunrise. 

Korschelt u. Heider (93, p. 1267) state that in most cases among the 
Ascidians self-fertilization appears to be prevented by the ripening of the 


206 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


male and female sexual elements at different periods ; although in some 


cases, where the sexual products mature simultaneously, self-fertilization — 


is not excluded. Neither of these statements holds good for Ciona. 
Although in the adult period it produces both sexual elements through- 
out the spawning season, and discharges them simultaneously, self-fer- 
tilization rarely occurs, — a conclusion to which I have been led by 
repeated experiments. The most complete series of these experiments 
will be briefly described. 

The observation had been made that an individual accidentally left 
overnight in an aquarium by itself laid eggs which failed to develop ; 
whereas, when two or more individuals were placed together in an aqua- 
rium, all other conditions being the same, the eggs laid developed almost 
without exception. 

First Experiment. 

Acting on the suggestion thus offered, I placed together in an aquarium 
two or three small, clear individuals (evidently young) ; in another aqua- 
rium was placed an equal number of very large-sized (old) individuals. 
A greater number of eggs was laid by the large individuals, as one would 
naturally expect, but the eggs in both aquaria were perfectly fertile. 
This experiment showed that ripe eggs and sperm are produced both 
by young and by old individuals. 


Second Experiment. 


Twenty rather large-sized individuals were selected for experimentation 
and divided into two lots, A and B, of ten individuals each. The animals 
of each lot were carefully washed and placed in clean glass dishes filled 
with fresh sea-water. The individuals of lot A (Table I.) were placed 
each in a separate dish, those of lot B (Table II.) were placed two in a 
dish. The next morning a careful examination of each aquarium was 
made to determine what proportion of the eggs laid had been fertilized. 
The experiment was repeated on five successive days ; on the sixth day, 
as a control experiment, the lots were interchanged, the animals of lot 
A (Table II.) being paired, and those of lot B (Table 1.) isolated. The 
results for the six days are embodied in Tables I. and II. 

Taking an average of the fifty-eight cases in which eggs were laid by 
isolated individuals (Table I.), we find that 4.8% of the eggs were fertilized. 
The occurrence on a single day in two cases of fertilization of 90% of the 
egos laid makes me suspect that the dishes were not properly cleaned 
on that day, and that live spermatozoa may have remained clinging to 
the sides of the dish after the previous day’s experiment. If so, and if 


i il er 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 


TABLE I.—Cuose FertixizarTion. 
Lot A. 


Day. 


90%, fertilized 


25% fertilized 


10% fertilized 


5% fertilized 


4%, fertilized 


None fertilized 


No eggs laid 


Total 


Average fertility = 4.8%. 


TABLE II.— Cross FEerrivizaTIon. 
Lot B. Lot A. 


Day. 


100% fertilized | 5 4 4 5 5 5 28 


20% fertilized 1 1 


None fertilized 1 1 


Total 


Average fertility = 94%. 


207 


aot 


208 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


the dishes were by chance interchanged, a certain amount of cross fertili- 
zation may of course have been possible. 

On the three succeeding days (fourth, fifth, and sixth) greater precau- 
tions were taken, and the jars were dried as well as washed before the — 
experiment was repeated. It will be observed that the proportion of — 
eges fertilized on those days was distinctly less than on the first three — 
days. ¥ 

Taking an average of the thirty cases in which eggs were laid in — 
aquaria containing each ¢wo individuals (Table Il.), we find that 94% — 
of the eggs laid were fertilized. 

The single instance in which none of the eggs laid in one aquarium — 
were fertilized may be explained by a failure on the part of one of the 
two animals confined together to emit the sexual products on that par- 
ticular occasion. Table I. indicates that such cases sometimes occur ; 
for in two instances out of sixty no eggs at all were laid. 

Comparing the results of the two tables, we see that under conditions 
allowing of only close (self-) fertilization (Table I.), less than 5% of the 
eggs developed ; whereas under conditions permitting of cross fertil'za- 
tion (Table II.) at least 90% of the eggs developed. 

The question now arose, Do eggs laid by isolated individuals fail to 
develop because the parent does not discharge sperm at the proper time 
(perhaps for want of stimulation by another individual), or do the eggs 
fail to develop because they are ¢ncapable of fertilization by sperm from 
the same parent? To settle this point if possible, resort was had to arti- 
ficial fertilization. 

Third Experiment. 

The same animals employed in the second experiment were also used 
in this one. Half of the individuals of each lot were taken for an attempt 
at close fertilization, the other half being reserved for an attempt at 
cross fertilization. Each animal was dipped in 90% alcohol to kill any 
spermatozoa which might be adhering to it ; the fingers and instruments 
used were treated in the same way. Eggs and sperm were removed 
from the sexual ducts of the animal, and thoroughly mixed in a dish of 
clean fresh sea-water, the dish having been previously carefully washed 
and then dried. 

The second ten were treated in exactly the same way, except that the 
sexual products — both male and female — of two individuals were mixed 
together in a single dish. 

The proportion of fertilized eggs in each dish was subsequently care- 
fully observed. The results are given in Tables III. and IV. 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 209 


TABLE II].—Artirici1aL CLosE FERTILIZATION. 


50 % fertilized 
4% Average for ten cases, 6% = proportion of 


1% egos fertilized. 
1% gs 


None 


100 % fertilized Average for five cases, 90% = proportion of 
50% “ eggs fertilized. 
Total 


As the animals employed in the above experiment had been confined 
in the laboratory for some days, and the production of the sexual elements 
had in consequence considerably diminished, it was thought desirable to 
repeat the experiment on animals freshly collected. This accordingly 
was done with the following equally conclusive results. 


TABLE III. a.— Artiricirat CLose FERTILIZATION. 


50% fertilized 


1213 “c : 
a « Average for ten cases, 9.4% = proportion of 


5% “ eggs fertilized. 
:, 
None “ 

Total 


TABLE IV.a.— Artiric1aAL Cross FERTILIZATION. 


100% fertilized in every case! 


210 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


The proportion of close fertilized eggs was greater in this experiment 
than in the preceding. Many of the eggs so fertilized, however, never 
developed beyond the 2-or 4-cell stage. The cross fertilized eggs all 
developed normally and at the same rate. 

Combining the results of Tables III. and III. a, and those of IV. and 
IV. a, we get an average of 7.7% of the eggs developing after close fer- 
tilization, and 95% developing after cross fertilization. These averages 
agree fairly well with those obtained from Experiment 2, which were 
4.8% and 94% respectively. 

Experiment 3 shows conclusively that, in the case of Ciona, eggs are 
to a large extent incapable of fertilization by sperm from the same indi- 
vidual as the eggs. Cross fertilization must, therefore, be the rule, and 
close fertilization the exception under natural conditions. The rare 
occurrence of close fertilization is probably due to a lack of mutual 
attraction between eggs and sperm produced by the same individual, an 
attraction invariably existing between the eggs of one individual and 
the sperm of another, and probably chemical in its nature. This case 
is paralleled in certain flowering plants, whose pollen will not germinate 
when placed on the stigma of the flower from which it was taken, though 
on the stigma of other flowers of the same species of plant it germinates 
readily. 

There seems to be a particular time of day in the case of each species 
of simple Ascidian for the discharge of the sexual products. Different 
aquaria, in which are placed individuals of the same species, if they are 
subjected to the same conditions of temperature, etc., invariably contain 
eggs in exactly the same stage of development. ‘This shows conclusively 
that the time of egg-laying has been the same in the case of each 
aquarium. For, on account of the rapidity of development, a slight 
difference in the time of egg-laying would be readily detected by a dif- 
ference in the stage of development exhibited by the eggs in different 
aquaria. In the case of Ciona the sexual products are discharged about 
an hour or an hour and a half before sunrise. The stimulus to their 
discharge is probably the increasing light of daybreak. 

If at about the time mentioned one approaches the aquarium with a 
lighted lamp, he will see the animals suddenly contract violently two or 
three times in succession, then resume their accustomed tranquillity. A 
careful examination will then reveal the eggs floating as little golden 
specks in the thoroughly agitated water. Soon they begin to settle to 
the bottom of the aquarium and can then be collected in convenient 
quantities by means of a pipette. The violent expulsion of the contents 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 211 


of the atrium simultaneously with the release of the sexual products 
from their respective ducts, must secure under natural conditions a 
wider distribution and more thorough mixing of the eggs and sperma- 
tozoa than would otherwise occur.! 

The season of spawning of Ciona probably extends in this country, as 
in Europe, from spring to autumn. I have never collected adult speci- 
mens which did not contain mature eggs and spermatozoa, though I 
have taken them as early in the season as the 10th of June and as late 
as the 22d of September. 

The development of the ovum is very rapid, as I shall show further 
on, and the larval period brief. The growth of the metamorphosed 
individual must also be very rapid, as the following facts indicate. In the 
summer of 1892 specimens of Ciona were abundant in a certain locality 
at Newport. But the succeeding winter was a cold one, and seems to 
have killed off those individuals which were situated in very shallow 
water. In the summer of 1893 specimens were to be found only at a 
depth of over two feet below low-water mark. In August and Septem- 
ber of the next year, however, they occurred in abundance just below 
low-water mark. But those so situated were rather small, not exceed- 
ing 7 cm. in length, very clear, and free from dirt or parasitic growths, 
thus giving evident signs of youthfulness. They cannot have been over 
fifteen months old, and may have been much younger. Yet they were 

sexually mature, and produced eggs in abundance. 


1 The time of egg-laying is about the same — viz. just before daybreak — in the 
case of Molgula Manhattensis,.on which I made some observations in the United 
States Fish Commission Laboratory at Wood’s Holl, Mass., in June and July, 1894. 
Cynthia, whose habits I studied at the same place, lays its eggs with equal clock- 
like regularity, but toward nightfall instead of at daybreak. The late afternoon is 
also the time of spawning for Amphioxus (Wilson ’93, Willey 794). The manner of 
egg-laying is the same in Molgula as in Ciona. Herein my observations differ from 
those of Kingsley (83), who states that in Molgula fertilization occurs within the 
atrium, and that the eggs are for some time afterward retained there. I have 
never found embryos within the atrial chamber, though I have often seen them 
adhering to the bodies of the parent individuals, where some eggs had probably 
settled at the time of spawning. My observations regarding the manner of 
cleavage in M. Manhattensis also differ from those of Professor Kingsley. He 
states that the cleavage is unequal, much as in certain Mollusks, and results in 
the formation of a cap of very small micromeres resting on a few very large 
macromeres. According to repeated observations of my own, made both on 
naturally and on artificially fertilized eggs, the cleavage progresses very much 
as in other Ascidians, the first two cleavages being equal. I think Professor 
Kingsley must have been misled by appearances in immature eggs obtained by 
dissecting out the ovaries for artificial fertilization. 


bo 


12 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


Allusion has been made to the rapidity of development of the egg. 
Within twelve hours after fertilization the larval form is attained, the 
tail being coiled round the trunk within the egg membranes. Hatching 
usually occurs within the next twelve hours, i. e. in the first night after 
the laying of the eggs. It is brought about by twitchings of the larval 
tail, which finally rupture the egg membranes. Under certain con- 
ditions the larva does not succeed in breaking through the egg mem- 
branes. Metamorphosis then sets in almost immediately, and is 
completed within the egg membranes, a functionally free-swimming 
stage being wholly suppressed. ‘This is regularly the case in Molgula 
Manhattensis, where hatching of the larva is exceptional, the new, meta- 
morphosed individual arising just where the egg settled after it was 
thrown out into the water and fertilized. However, in Ciona the more 
primitive course of events is usually pursued. The larva then escapes 
from the egg membranes as a miniature tadpole, the ‘test cells” clinging 
to its thin and adherent covering of homogeneous, non-cellular mantle 
substance secreted by the ectoderm. These test cells are soon brushed 
off as the tadpole swims about ; they have no connection, as is now well 
known, with the cells to be found later in the mantle of the adult. 

The larve avoid the daylight and swim toward the least brightly 
illuminated side of the aquarium.’ Here they attach themselves, usually 
near the surface of the water, to the side of the aquarium. Sometimes 
the attachment is by the head end, as it is commonly said to be, but I 
have more often observed the larvee attached by the sticky mantle sub- 
stance at the tip of the tail, the body then hanging head downward 
against the side of the aquarium. 

The larval stage varies in duration from twenty-four hours to several 
days. It is terminated by the beginning of metamorphosis, whose suc- 
cessive steps are well known through the description of Kowalevsky (66 
and ’92), Willey (’93), and others. 


1 T have observed that the larve of Amareecium also avoid the daylight, i. e. 
are negatively phototactic ; but the larve of Botryllus are strongly positively pho- 
totactic, swarming toward ordinary daylight. This difference may perhaps be 
explained by the difference in habitat of the parent organisms. Botryllus, whose 
larve seek the light, is commonly found in well illuminated places, e. g. adhering 
to floating eel-grass. On the other hand, Ciona and Amarecium, whose larve 
avoid the light, more often occur in darkened places, the former on the under side 
of stones, the latter adhering to piles underneath wharves, or on the sea bottom in 
sheltered spots near shore. 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 213 


III. METHODS. 


1. Killing, Preservation. 


Whenever it was desired to kill a lot of eggs, a sufficient quantity of 
them was collected in a pipette from the bottom of an aquarium and 
transferred to a watch-glass, or directly to a small vial of two drams’ ca- 
pacity, in which the eggs were ultimately stored. After the eggs had 
settled to the bottom of the dish, the water was carefully removed and 
the killing reagent applied. 

The eggs were ultimately preserved in 90% alcohol, and the vials 
tightly corked, or preferably stoppered with cotton plugs and stored in 
tightly sealing glass jars. When the latter method is employed, the jars 
must be kept right side up in transportation, otherwise the small eggs 
will settle into the cotton plugs and be lost. However, the extra trouble 
which this method necessitates is well worth taking, for it entirely avoids 
the injurious effects on preserved material sometimes caused by the 
tannin which alcohol will extract from corks, if they are used. 

Several killing reagents were employed, viz. Flemming’s fluid, Her- 
mann’s fluid, picro-nitric, corrosive-acetic, and Perenyi’s fluid. The 
blackening effects of the first two reagents made material killed in them 
unfit for use in the study of eggs as whole objects. Likewise in the case 
of sections the results from them were disappointing. The only real 
service rendered by either of these two reagents was in demonstrating in 
the egg by their blackening effects the character and distribution of the 
fatty yolk granules. Most serviceable of all the reagents employed on 

_the eggs and embryos up to the period of hatching was Perenyi’s fluid. 
It renders the abundant yolk clear and transparent, and preserves all 
structures perfectly, without distortion by either swelling or shrinking. 
Its use does not in my experience interfere in the least with sharp differ- 
ential staining. The fluid was allowed to act for about twenty minutes, 

_ then followed by 70% alcohol, which, to insure removal of every trace of 
the killing reagent, was changed once or twice in the course of the next 

i twenty-four hours, and replaced at the end of that time with 90% alco- 

hol. A longer treatment with the killing reagent, extending to three or 
four hours, seemed to give no added advantage, but to interfere slightly 
with subsequent staining. 

Picro-nitric also gave good results, but for the pre-larval stages not so 

good as Perenyi’s fluid, its clearing effects being less. It seems, however, 


1 For the composition of the killing reagents and stains mentioned in this paper, 
see Lee’s ‘The Microtomist’s Vade Mecum,” 3d edition, London, 1893. 


214 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


to have been for the larval stages the best reagent which I employed. 
Davidoff’s corrosive-acetic mixture, which has been much used of late by 
workers on ascidian embryology, is in my experience less faithful in its 
preservation than Perenyi’s fluid, for it shows a tendency to swell certain 
structures, and lacks the instantaneous hardening effects of that reagent. 


2. Decortication, Staining, Mounting. 


The egg of Ciona is surrounded by a series of egg membranes, a cor- — 
rect idea of which is given by the figure of the mature egg of Ascidia 2 
canina, reproduced after Kupffer (’72) in Korschelt u. Heider’s “ Lehr- — 
buch d. vergl. Entwickluugsgeschichte,” Figure 736. The egg cell is 
seen to be surrounded by a clear space — probably occupied by jelly — ; 
bounded by the test cells, which are arranged in a rather compact layer 
one cell deep, so that they seem almost to form an epithelium under- 
neath the chorion. The chorion is a structureless transparent mem- 
brane, upon which, as on a basement membrane, the follicle cells 
(‘‘Schaumzellen’’) rest. In the egg of Ciona, after it is thrown out 
into the water, these highly vacuolated cells are even more conspicuous — 
than in the egg of Ascidia as figured by Kupffer. They extend out 
radially about twice as far as indicated by Kupffer’s figure, forming a 
sort of halo round the egg. The highly refractive nuclei are carried out 
to the pointed outer ends of the tapering follicle cells. 

The presence of the follicle cells and test cells did not interfere 
seriously with the study of the early stages of cleavage in the living egg, 
since the clear space between the egg cell and the layer of test cells 
allows one, with a sufficiently strong illumination, to make out perfectly 
the outline of the blastomeres and sometimes even nuclear figures in 
them. But upon preservation in alcohol the envelope formed by the 
test cells, chorion, and follicle cells collapses, obliterating the clear space 
and becoming closely applied against the egg cell, thus forming a very 
serious obstacle to the study of the egg asa whole object. This obstacle 
I was able to remove by following in a modified form a very ingenious 
method devised by Chabry (’87, p. 169) for the removal of the follicle 
cells from the living egg of Ascidiella, a process which he called “ decor-_ 
tication.” It consisted in simply sucking the eggs into a fine capillary 
glass tube too small to admit the eggs without the removal of their 
follicle cells, yet large enough to allow the passage uninjured of the egg 
itself. 

In applying this method to preserved material, I first stained the eggs, 
as a rule, so that they might be more easily seen. Upon transferring 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 215 


them to alcohol of a low grade, or to water, the egg envelopes would 
again stand out clear of the ovum, as in the living egg. By then suck- 
ing the eggs one at a time into a glass tube of the proper calibre, the 
entire envelope, consisting of follicle cells, test cells, and chorion, could 
_be removed with considerable facility, and in the majority of cases with- 
out injury to the egg itself. Eggs thus decorticated and then mounted 
afforded excellent surface views. 
The eggs are rather opaque, on account of the large amount of yolk 


— 


ae. 


which they contain, so that any stain except a very faint one is an ob- 
stacle in the study of whole preparations. Excellent results were obtained 
iW mounting in balsam, without any staining whatever, eggs which had 
_ been killed in Perenyi’s fluid and decorticated.* But for the 64-cell and 
later stages staining was found desirable. Many carmine and hematoxy- 
lin stains were tried ; the one which gave by far the best results being 
Orth’s picro-carminate of lithium. The eggs were treated with a small 
~ amount of this stain in a watch- glass for from six to twenty-four hours, 
then washed thoroughly in water. By this method resting nuclei are 
a 


al 


aoe 


stained bright rose-color, while all other structures take only a faint 
yellow color from the picric acid, and give up even this if the washing is 
sufficiently prolonged. But the carmine stain in the nuclei is extremely 
tenacious, and does not fade in the least upon prolonged washing in water 
or preservation for months in strong alcohol. After the eggs had been 
stained and decorticated, they were dehydrated, cleared in xylol or cedar 
oil, then mounted in balsam, the cover glass being supported with small 
_ glass rollers made from fine capillary tubes. These served the double pur- 
_ pose of preventing the crushing of the egg and allowing it to be rolled 
into any desired position by movement of the cover glass. Changing the 
position of the egg, however, is not often necessary, for at an early stage 
it takes on a flattened form, which causes it to come to rest with the dorsal 
or the ventral surface uppermost. This is the case at all periods between | 
the 24-cell stage and that at which the neural tube begins to close, ex- 
cept for a brief period, when the embryo consists of from forty-six to 
sixty-four cells, and the vertical axis becomes equal to or even greater 
than the longitudinal axis. Then there is no single position of stable 
repose for the embryo, and rolling is often easel to bring it into the 
positions desired. 
The self-orientation of the egg during most of the early stages was of 


1 JT find that Lillie (95) has obtained good results in the case of the eggs of the 
mollusk Unio by mounting, without staining, material killed in Perenyi’s fluid. 
_ He, however, used glycerine instead of balsam as a mounting medium. 


216 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


great service in sectioning. When this was desired, the egg, previously 
studied as a whole object, was returned to xylol. The transfer was accom- 
plished by placing the slide on which it was mounted in a shallow por- 
celain dish containing a little xylol. This soon dissolved away the 
balsam, and left the egg free and clearly visible against the white back- 
ground. The egg was next removed to a shallow watch-glass with a 
perfectly flat bottom, which was previously smeared with a thin layer 
of glycerine. Any superfluous xylol was removed from about the egg 
with filter paper, and a small amount of melted paraffine poured over it, 
enough to fill the watch-glass to a depth of 3 to 5mm. The whole was 
then set over the paraffine bath for fifteen or twenty minutes, when it 
was placed floating on a dish of water to cool. This being accom- 
plished, the parafiine block was removed from the watch-glass, and the 
egg, which of course had settled to the bottom and lay with its long axis 
parallel to the surface of the block, was oriented under the compound 
microscope in any manner desired. The thinness of the block generally 
allowed plenty of light to pass through it for this purpose, and it was 
usually not difficult, owing to the shape of the embryo, to determine its 
axes. Sections were usually cut 62 p in thickness. 

The staining which was found most advantageous for the study of the 
egg as a whole object was altogether too faint for sections. These were 
accordingly given a further staining after fixation to the slide. Ehrlich’s 
hematoxylin was employed, diluted one half with water. After immer- 
sion in the stain for from twenty minutes to an hour, the sections were 
washed in water to remove the superfluous stain, then to decolorize were 
placed in 35% alcohol containing 0.1% hydrochloric acid. Here they were 
allowed to remain until quite pale in color, usually for about five min- 
utes. They were then rinsed in 35% alcohol and held for an instant over 
the unstoppered mouth of an ammonia bottle, a treatment which gave 
the hzmatoxylin remaining in the sections a deep blue color, and in- 
sured the permanency of the stain. The sections were then passed 
through the grades of alcohol, cleared in xylol, and mounted in balsam. 
This process, when properly conducted, resulted in a beautiful and sharply 
differential double stain. The nuclei retained the light rose tint given 
them by the carminate of lithium, for the superadded hematoxylin stain 
had been entirely removed from them, except in the chromatic elements, 
which possessed a deep black color. Cell boundaries, attraction spheres, 
and other cytoplasmic structures, were clearly brought out, and the fun- 
daments of various organs, as, for example, chorda, mesoderm, and defini- 
tive endoderm, were distinguished one from another with great sharpness 


’ 
CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 217 


by the different tints of blue which they exhibited. Iron hematoxylin 
was sometimes employed instead of Ehrlich’s, but the results were no 
better — indeed not so good — for the differentiation of organs or their 
fundaments. 

For studying the processes of maturation and fertilization sections 
alone could be employed on account of the opacity of the eggs. In mak- 
ing sections of these stages orientation was of course impossible, so that 
a large number of the eggs was embedded together, without previous 
decortication, and cut at random. The egg membranes, so far from 
being an obstacle, were at these stages a positive advantage, since they 
served to protect and hold the polar globules in place. The material 
employed in the study of maturation and fertilization stages was killed 
either in Perenyi’s or in Hermann’s fluid, the best results being obtained 
from the former. For convenience the killing of each day will be referred 
to as a series (A, B, or C), made up of lots (1, 2, 3, etc.) which were 
killed at intervals of about ten minutes, the first lot being killed as soon 
after the laying as a sufficient number of eggs could be collected, usually 
about five or ten minutes. 


IV. MATURATION AND FERTILIZATION. 


The eggs of series A, lot 1, show an early stage in the process of matu- 
ration, namely, the formation of the first polar globule. Figure 1 repre- 
sents a section through one of the eggs of this lot most advanced in 
development. The egg envelopes, which rest close down upon the egg, 
are left out in this and all the other figures. Already at this stage we 
recognize that the egg is made up of two unlike hemispheres, one richer 
in yolk, the other richer in protoplasm. The former occupies the future 
dorsal or endodermal side of the egg, and at the centre of its surface, as 
stated in my preliminary communication (’94), the polar globules form. 
The cell division which will give rise to the first polar globule is seen in 
this figure to be already well advanced, the chromatin being accumulated 
at the two ends of the spindle. About the deeper end of the spindle there 
is a small space free from yolk granules and occupied by a finely granular 
deeply staining mass of protoplasm, of which we shall have more to say. 
The entire remainder of the dorsal hemisphere, except that small portion 
of it occupied by the spindle itself, is filled with rounded yolk granules 
(ef. Fig. 2) of a rather uniform size, closely packed together, but with 
slender films of staining protoplasm passing between and around them. 
Davidoff’s (’89) beautiful figures, hat his Tafel VI. Fig. 33, 


VOL. XXVII. — NO. 7. 


218 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


give a correct idea of this “Schaumwerk ” structure, if one imagines the 
yolk granules many times smaller and the protoplasmic films much more 
slender than in the egg of Distaplia as represented by Davidoff. 

The ventral hemisphere also is filled with yolk granules, but here the 
protoplasmic packing between them is more abundant and less uniformly 
distributed. It is most conspicuous at the surface, where it forms a 
thin layer nearly free from yolk granules spreading over almost the 
whole hemisphere. Within this layer it fades away gradually, but 
often, as in the case figured (Fig. 1), again becomes prominent at a little 
deeper level as a series of irregular blotches among the yolk granules ; 
then it once more grows fainter toward the centre of the egg, attaining 
the condition described for the dorsal hemisphere. 

The presence of a spermatozodn cannot be detected in the eggs of this 
lot. In those of Series B, lot 3, however, its influence is clearly visible. 
(See Figs. 2 and 4.) About fifteen minutes is estimated to have 
elapsed between the stage just discussed and the one here presented. 
At this stage we see in the ventral hemisphere, at some point just 
beneath the surface, a spherical region entirely free from yolk granules. 
(See Fig. 2.) Its central portion is occupied by a finely granular sub- 
stance, which stains in hematoxylin an intense blue, shading off some- 
what gradually into the more faintly and lightly colored protoplasm 
occupying the outer portion of the area and continuous with the sim- 
ilarly stained films of the Schaumwerk. At one point the yolk-free re- 
gion extends out to the surface of the egg. This probably represents the 
place of entrance of the spermatozodn, which we have reason to believe 
produces the clear area. The deeply staining substance at the centre of 
this area is the male archoplasm or attraction sphere. It is undoubtedly 
similar in nature, as it is in optical appearance, to the darkly stained 
substance seen at the deep end of the maturation spindle in Figure 1, and 
which may therefore be called the female archoplasm. The male pro- 
nucleus cannot be made out in the egg a portion of which is shown in 
Figure 2. In other eggs of the same lot, however, it can be clearly seen ; 
for example, in Figure 4, which represents a stage a little more advanced 
than the one seen in Figure 2. The area free from yolk is seen in Figure 4 
to have enlarged somewhat; the attractive influence of the archoplasm 
at its centre has manifestly-been extended over the greater portion of the 
hemisphere in which it lies. This fact is indicated diagrammatically by 
the dotted lines in the figure. They are meant simply to indicate that 
those films of the protoplasmic Schaumwerk which run radially with 
reference to the attraction sphere have become thicker and more promi- 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 219 


nent than those running in other directions. Along them as radii 
doubtless protoplasm is passing to augment the yolk-free area. Nothing 
in the nature of “fibres” has been observed in them. Excentrically 
situated in the yolk-free area (Fig. 4) is seen the male pronucleus, a 
perfectly clear oval body, with a delicate but sharp boundary. Its long 
axis lies radially with reference to the attraction sphere, which mani- 
festly exerts on it a directive influence. Figure 4 represents the eleventh 
of a series of eighteen sections. The sixteenth section of the series is 
shown in Figure 3. It contains the second maturation spindle, at either 
end of which is an attraction sphere in the centre of a slight accumula- 
tion of protoplasm. The chromosomes cannot be clearly made out, but 
perhaps lie aggregated in a small dark mass close down against the 
attraction spheres. It is evident that the amount of chromatin in- 
volved in this division is less than in the case of the first maturation 
division (cf. Fig. 1). The obliquity of the plane of sectioning to the 
dorso-ventral axis of the egg makes this spindle appear to lie quite a 
little below the surface of the egg. Such, however, is not the case; it 
comes close up to the surface, but obliquely, not vertically, as did the 
first maturation spindle. Indeed, an examination of other specimens, 
less advanced, shows that it first appears in a horizontal position, i. e. 
at right angles to the direction of the first maturation spindle as seen in 
Figure 1, but later rotates so that one end of the spindle lies deeper in 
the egg than the other. The first polar globule does not really lie in 
this section, but has been projected there from its real position oh the 
margin of the next section, the seventeenth of the series. 

In Figure 5 is represented a section, the fifth of a series of sixteen, 
through an egg of Series A, lot 3, killed twenty minutes later than 
lot 1 of the same series (cf. Fig. 1). The section passes obliquely in a 
dorso-ventral direction, unlike those shown in Figures 2-4, which were 
more nearly horizontal. On the ventral margin of the section is seen the 
cap of protoplasm which as early at least as the beginning of maturation 
covered that side of the egg. The male archoplasm has moved deeper 
into the egg, and its attractive influence has been extended so that it is 
now manifested over the greater portion of the section. In consequence 
of this attraction on the protoplasm the area free from yolk has con- 


1 A rotation of the maturation spindles from an original tangential to a radial 
position has been observed repeatedly in other animals; in the case of the second 
spindle, the tangential position is doubtless correlated with the derivation of its two 
archoplasmic masses from the single archoplasmic mass left in the egg after the 
completion of the first maturation division. 


220 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


siderably enlarged. The male pronucleus has also increased in size and — 
followed the lead of its attraction sphere toward the centre of the egg. 
In the dorsal half of the section is seen the female pronucleus, already 
grown to considerable size. In it can be discerned small chromatic 
granules, and behind it and deeper in the section the female archoplasm. 
This archoplasm seems to be much less energetic than that of the male 
element, for its influence is scarcely perceptible, even on the portion of. 
the egg in which it lies, and it does not appear to modify either the shape 
or course of the female pronucleus, which, as we shall see, moves toward 
the male archoplasm leaving its own behind. The polar globules repre- 
sented at the margin of this section do not as a matter of fact occur in 
that position, but at the margin of the preceding section. If that sec- 
tion were properly superposed on this, the polar globules would lie over, 
but a little to the left of the female pronucleus. 

A stage semewhat later than the one just described, though found in 
the same lot of eggs, is shown in Plate II. Figures 7-10, which represent 
the fourth, seventh, tenth, and twelfth sections respectively of a series 
of sixteen. In Figure 7 is seen the male pronucleus with its archoplasm 
now divided ; in Figure 8, the female pronucleus ; in Figure 9, the female 
archoplasm ; and in Figure 10, the polar globules marking both the 
centre of the future dorsal surface of the embryo, and the point from 
which the female pronucleus starts in its journey through the egg toward 
the male pronucleus. The position of these various bodies with relation 
to one another can be most clearly illustrated by two reconstructions 
(Figs. 11 and 12) upon planes perpendicular to the plane of sectioning 
and at right angles to each other. Suppose the sections piled one above 
another in their original order and position, the first section of the series 
being uppermost and the egg thus reconstructed to be viewed as a trans- 
parent object in the direction of the arrow at the left of Figure 7. One 
would then see the appearance shown in Figure 11, which is a projection 
of the egg and the most important bodies in it upon a plane parallel to 
the line ab (Fig. 7), and perpendicular to the plane of Figure 7. 

If the egg be viewed in the direction of the arrow at the top of 
Figure 7, one gets the appearance shown in Figure 12, which is a pro- 
jection upon a plane parallel with the line a’ 0’ in Figure 7, and perpen- 
dicular to the plane of that figure. 

A comparison of the stage under discussion with that represented in 
Figure 5 shows that considerable changes have occurred in the interval 
between them. The male pronucleus (Fig. 7) has grown to much 
greater size and contains several conspicuous chromatic granules. In- 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. yA 


stead of a siugle attraction sphere, there are two, both well defined and 
at a considerable distance apart. An examination of other eggs of the 
same lot shows how the condition here existing has come about. The 
male archoplasm moving in advance of its pronucleus (cf. Fig. 5) has 
gradually eiongated transversely to its line of progress, arranged itself 
about two centres instead of one, and finally constricted itself into two 


‘distinct spherical masses, which move apart, and by their combined 


action on the male pronucleus draw it forward to a position midway 
between them, so that its long axis lies in the line joining their centres. 
The female pronucleus (Fig. 8) has approached to within a short dis- 
tance (about one fifth the diameter of the egg) of the male pronucleus. 

It has grown to an equal size with the male pronucleus, and, like 
it, contains large chromatic granules. No trace of an archoplasmic 
body can be seen in connection with it, nor in either of the adjacent 
sections. However, what are unmistakably the remains of one are visi- 
ble three sections behind the female pronucleus. (See Fig. 9; compare 
also Figs. 11 and 12.) This archoplasmic body shows signs of disinte- 
gration, being rather diffuse and exerting apparently no attractive 
influence on the egg protoplasm. The female pronucleus has clearly 
passed beyond its control, and is now advancing rapidly to unite with 
the male pronucleus. One might doubt that the body described is 
identical with a female archoplasm, were it not perfectly constant in its 
appearance at this stage behind the female pronucleus in the path of the 
latter from the point where the polar globules were formed toward the male 
pronucleus. Moreover, though diligent search has been made, a similar 
body has never been found at this stage in any other portion of the 
ovum, except in connection with the male pronucleus. 

In from five to ten minutes after the stage just described the two pro- 
nuclei are seen to have come together (Plate III. Fig. 13, and Plate I. 
Fig. 6). They are indistinguishable from each other so far as size and 
optical appearance are concerned, and are flattened against each other, 
but their nuclear membranes remain intact, and there is no mingling of 
their substance until the first cleavage is about to take place. (See 
Plate III. Fig. 14.) At the stage shown in Plate II. Fig. 7, we saw that 
the male pronucleus was already elongated between its two attraction 
spheres. The female pronucleus is seen in Figure 13 (Plate III.) to have 
joined it while it is still in that condition. Both have further increased 
jn size. Very soon the nuclear membranes disappear, the attraction 
spheres move farther apart (cf. Figs. 13 and 14, Plate IIT.), and a spindle 
forms between them, on whose equator are seen the chromosomes. 


222, BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


To recapitulate. Jn the impregnated egg of Ciona two archoplasmic 
masses can be recognized, one in connection with each of the pronuclei. 
That derived from the spermatazodn is much the more energetic of the two, 
and is alone concerned in bringing the pronuclet together. While the pro- 
nuclei are still a considerable distance apart, the male archoplasm divides 
into two distinct attraction spheres, between which the first cleavage spindle 
later forms. The female archoplasm degenerates, taking no part whatever 
in the formation of the first cleavage spindle. There is accordingly in the 
fertilization of Ciona no union of male and female archoplasms. 

Let us compare briefly these conclusions with those of other recent 
observers on the subject of the attraction sphere in fertilization. 

The fertilization of the Tunicate egg has been studied hitherto by 
Boveri (90) and Julin (’93). Boveri’s observations, made on Ciona 
intestinalis and Ascidia mentula, were, as he states, incomplete on 
account of an accident to his preserved material. It was his opinion 
that no astral radiations (“ Polstrahlungen’”’) are present in the matura- 
tion of the egg, and that the two asters of the first cleavage spindle are 
derived by division from a single one arising in connection with the 
spermatazodn soon after its entrance into the egg. Julin was able to 
confirm on Styelopsis grossularia the observations of Boveri, and to sup- 
plement them, as he says, by demonstrating at the centre of each aster 
of the first cleavage spindle a centrosome. No figures, however, accom- 
pany Julin’s paper; moreover, he states that his observations were 
restricted to two stages, corresponding to those shown in Boveri’s 
Tafel XII. Figs. 27 and 29. 

Though my own conclusions are in entire agreement with those of 
Boveri and Julin as to the derivation of the attraction spheres of the 
first cleavage spindle exclusively from the spermatazodn, my observations 
differ from theirs regarding certain minor points, as the reader may 
learn by consulting the papers cited. 

On the subject of fertilization in groups of animals other than the 
Tunicata there is an enormous literature. I shall refer to only a few of 
the most recent papers. 

In 1891 Fol described the famous “ quadrille of the centres”’ as occur- 
ring in the fertilization of the sea-urchin egg. According to his account, 
there arises in the egg from the tip of the spermatazoodn, a centre of 
attraction (‘‘spermocentre”), which later divides. In connection with 
the egg nucleus appears another centre of attraction (‘‘ovocentre”’), 
which likewise divides. Upon the meeting of the pronuclei, each half- 
spermocentre unites with a half-ovocentre to form an astrocentre. The 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. Wea 


two astrocentres arise on opposite sides of the cleavage nucleus, and 
between them the first cleavage spindle forms. 

A short time after the publication of Fol’s paper, Guignard (’91) 
described as occurring in the fertilization of a flowering plant a similar 
union of male and female centres of attraction (“sphéres directrices”). 
More recently Conklin (94) has observed its occurrence in the case of a 
mollusk, Crepidula. 

*Fol’s observations, however, are flatly contradicted by the careful 
studies of Wilson and Mathews (’95) on three different genera of Kchi- 
noderms. They find that ‘the central archoplasm sphere (‘ attraction 
sphere’) of the cleavage amphiaster is derived by direct and unbroken 
descent from the central mass of the sperm-aster without visible partici- 
pation of an egg-aster.” 

Fick (’93) also observed that in the fertilization of a Vertebrate, Axo- 
lotyl, the centrosomes of the first cleavage spindle are derived exclu- 
sively from the spermatozodn. Brauer (’92) arrived at a similar 
conclusion regarding the fertilization of a crustacean, Branchipus, and 
Mead (’95) regarding a worm, Cheetopteris. These observations are in 
entire agreement with those made prior to Fol’s announcement of the 
“Quadrille” by Boveri (’88) on Ascaris and Sagitta, and by Vejdovsky 
(88) on Rhynchelmis. Boehm (’88) had also expressed with some 
caution a similar view regarding Petromyzon. 

On the other hand, Wheeler (95), in a paper published simultane- 
ously both with that of Wilson and Mathews and with that of Mead, 
states that in Myzostoma both centres of attraction arise in connection 
with the egg nucleus, none whatever being produced by the spermatozoon. 


Summary on Maturation and Fertilization. 


(1) In a majority of the animals in which fertilization has been most 
recently studied the attraction centres of the first cleavage spindle are de- 
rived from the spermatozodn and from the spermatozoon only, 

(2) But in the fertilization of at least one animal, and undoubtedly in 
all cases of parthenogenetic development, the attraction centres arise solely 
in connection with the egg nucleus. . 

(3) Both these facts prove conclusively that the archoplasm, or “ organ 
of division,” is not a bearer of heredity, since in fertilization it may be 
derived from the sexual product of one parent only, whereas it is a well 
recognized law that heritable substance is contributed to the offspring by 
both parents equally. 

(4) If the archoplasm is furnished in some cases by the sperm only 


apg BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


and in others by the ovum only, it is not inconceivable that in yet other 
cases both may contribute to its formation. Therefore the observations 
of Guignard and Conklin are not necessarily irreconcilable with those 
more recently made by Wilson and Mathews, Mead, Wheeler, and my- 
self, as well as the earlier observations of others. In any case, however, 
the theoretical conclusions based on Fol’s “quadrille,” as to the share 
which the attraction centres enjoy in the phenomena of heredity, may 
now be definitely set aside.? 


V. POLARITY OF THE EGG. 


Attention has already been called to the fact that even before fertiliza- 


tion one axis of the egg, the vertical, has been determined. The point 


where the polar globules form is its dorsal pole, which lies at the centre 
of the surface of the less richly protoplasmic hemisphere. At some point 
on the surface of the opposite hemisphere, the spermatazo6n usually enters 
the egg, and there is reason to believe that its point of entrance deter- 
mines the median plane of the embryo, and so its antero-posterior axis. 
After the two pronuclei have met, they move toward the centre of 
the egg, and in that region the first cleavage spindle arises (Plate III. 
Fig. 14). It invariably lies parallel to a tangent at the point of forma- 
tion of the polar globules. The first cleavage plane, which in accord- 
ance with a general law is perpendicular to the spindle at its equator, 
passes through the point where the pclar globules arose and divides the 
ege into two equal blastomeres (Plate III. Fig. 15; cf. Plate V. Fig. 27). 


1 Boveri (95), in a paper recently received, completely confirms the observations 
of Wilson and Mathews regarding the source of the attractive bodies of the first 
cleavage spindle of the sea-urchin egg. He for the first time in his published writ- 
ings, so far as I know, gives a formal definition of the centrosome, applying the 
term to what Wilson and Mathews call the “archoplasm.” Boveri, if I rightly 
understand him, recognizes an archoplasm surrounding the centrosome, at least at 
certain stages, and specifically different both from the centrosome and from the 
general cytoplasm. 

What in the foregoing pages I have called indifferently archoplasm and attraction 
sphere undoubtedly corresponds with what Boveri in his latest paper (’95) defines 
as the centrosome. A centrosome in the sense of Heidenhain, that is,a simple, dis- 
tinct granule staining black in iron-hematoxylin, I have not been able to detect in 
the egg of Ciona; nor have I observed a substance (Boveri’s archoplasm) specifi- 
cally distinct from the egg cytoplasm, enveloping the attractive body (Boveri’s 
centrosome). As the reader will glean from the earlier pages of this chapter, I 
regard the substance forming the radiations about the attractive body as identical 
with the egg cytoplasm. — June, 1895. 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 225 


The section seen in Figure 15 shows that cleavage has progressed more 
rapidly from the ventral than from the dorsal surface. This is to be 
explained by the richer supply of protoplasm on the ventral surface. 

A study by reconstruction or otherwise of a series of sections through 
an egg in this stage invariably shows that cleavage has also progressed 
with unequal rapidity from the two ends of the embryo. 

That end at which cleavage is more advanced is destined to become 
the posterior end. In this case also the inequality in rate of cleavage 
is attended (probably caused) by an inequality in the distribution of 
protoplasm. The protoplasmic cap of the ventral hemisphere is always 
thicker at the future posterior end of the embryo than at the anterior 
end, and as the first cleavage plane cuts the egg, this accumulation of 


oD? 


protoplasm migrates in between the two blastomeres, its presence prob- 


ably being the accelerating force in the separation of the blastomeres. 

After the first cleavage is completed, the protoplasm, which had mi- 
grated in between the blastomeres, again returns to the surface and takes 
up a very definite position on the adjacent faces of the blastomeres just 
below the equator of the egg. (See Plate III. Fig. 17, x.) 

This region appears in the living egg as a clear area, and marks the 
spot where arise later the small flattened posterior cells found so useful 
in orientation by Van Beneden et Julin and others. That this clear 
area vs the region of their formation I have been able to establish by 
continuous observations of the living egg, controlled and completely sup- 
ported by the study of preparations. The thickened spot in the protoplas- 
mic cap of the ventral hemisphere at the beginning of cleavage, which seems 
to determine the posterior end of the embryo, J believe to be caused by the 
entrance of the spermatozodn. It is evident that the spermatazoén, 
unless it enters exactly at the ventral pole of the vertical axis, must lie 
upon entrance nearer to one end of the egg than to the other, supposing 
that it is in the median plane and ventral hemisphere of the embryo. 

The nearer end, I believe, becomes the posterior end of the embryo, 
and is determined for that fate by the accumulation of protoplasm in 
the region of entrance of the spermatazoén. It is impossible to say in 
any particular case exactly where the spermatazoén has entered the egg, 
for its presence there cannot be detected until it has begun to form a 
yolk-free area in the egg. However, I have never observed a case in 
which the spermatozo6n did not give evidence from its position of having 
entered the egg excentrically with reference to the lower pole of the ver- 
tical axis. Hence I conclude that cases of entrance at that pole, if they 
occur, are extremely rare. 


226 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


Summary on Polarity of the Egg. 


(1) The dorso-ventral axis of the embryo is predetermined in the egg 
before fertilization; the polar globules invariably form at its dorsal pole. 

(2) The spermatozodn may enter the egg at any point on its ventral 
hemisphere, that point probably determining, however, the median plane 
and posterior end of the embryo. 

(3) If we adopt the commonly employed terms animal and vegetative 
for the two poles of the unfertilized egg, we must call the ventral the 
animal pole, and the dorsal the vegetative pole. For it is the ventral 
half of the egg which contains a richer supply of protoplasm, and which 
consequently cleaves more rapidly and becomes the ectodermal side of 
the embryo; whereas the dorsal half of the egg contains less protoplasm, 
cleaves less rapidly, and forms the endodermal portion of the embryo. 

(4) We may say, accordingly, that the form changes accompanying ma- 
turation occur, in Ciona at least, and presumably in Ascidians in general, 
at the pole of the egg opposite to that at which they occur in Amphioxus, 
and, so far as known, in all other animals producing eggs with polar 
differentiation ; for the changes connected with maturation are uniformly 
reported to take place at the animal, i.e. at the more richly protoplas- 
mic pole, whereas in Ciona they take place at the vegetative pole. 


VI. CELL LINEAGE OF THE EMBRYO. 


The statement made in the preceding paragraph presents a condition 
of affairs so directly contrary to that found in other groups of animals, 
as well as to what has been assumed by all previous writers to be the 
case in Ascidians, that it requires ‘the presentation of unmistakable 
evidence in its support. Such evidence I have to offer, both from 
the study.of the living egg and from that of preparations. Before 
passing, however, to the consideration of this evidence, a word of 
explanation is necessary concerning the system of nomenclature to be 
employed. 


1. Nomenclature. 


In any extended work on cell lineage it is desirable to have some sys- 
tem of naming the individual cells which will indicate readily the exact 
history of each, — from what part of the matured ovum it has been 
derived, by how many divisions it is removed from the ovum, and from 
what other cells these divisions have separated it. In this paper I shall 


CASTLE: EMBRYOLOGY OF CIONA. INTESTINALIS. Oat 


follow with some modifications the system introduced by Kofoid (94) in 
his work on Limax. 

1. Each cell will be designated by a letter with two exponents. 

"2. The letter indicates the quadrant of the egg from which the cell in 
question has been derived, or in other words that cell of the 4-cell stage 
from which it is descended. Viewing the egg from the ventral or animal 
pole (the one opposite that at which the polar cells are formed), the left 
anterior quadrant is 4, the right anterior J, the right posterior C, and 
the left posterior D. In dorsal views, 4 and D are of course the right 
quadrants, and B and C the left. 

3. The first exponent indicates the generation to which a cell belongs ; 
that is, the number of cell divisions by which it is removed from the 
ovum. The ovum is generation one, the 2-cell stage two, the 4-cell stage 
three, etc. (See the Table of Cell Lineage on page 275.) 

4. The second exponent indicates the number of a cell in a generation, 
the cells of each quadrant being numbered independently from the animal 
toward the vegetative pole.’ If in any case two cells of common descent 
lie in an equatorial position, that one which is nearer the sagittal plane 
is given the lower numeral. 

To ascertain the designation of the mother cell of any particular cell, 
its first exponent must be diminished by one; and its second exponent, 
if an even number, must be divided by two, but if an odd number it 
must first be increased by one and then divided by two. 

In order to determine the daughter cell of a particular cell, simply 
reverse this process; that is, increase the first exponent by one, and 
double the second exponent. To determine the other daughter cell, 
diminish this second exponent by one. For example, the daughter cells 
of a®-* are a®8 and a®". 


2. Cleavage. 
A. Earzuy StTaGes or CLEAVAGE. 


(a) To 24-cell Stage. 


Figures 19-26 (Plate IV.) show eight views of a living egg, drawn by 
means of an Abbé camera lucida at successive stages, the egg remaining 
undisturbed in position under the microscope throughout the period of 
observation. The left side of the egg is, as I shall show, towards the 


1 Tn gastrulation, the cells about the vegetative pole are depressed to a lower level 
than the margin of the blastopore. In naming cells it is considered that the vege- 
tative pole is also depressed at that period, and lies constantly on the dorsal surface 
at the common point of meeting of the cells derived from the four quadrants. 


228 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


observer. In Figure 19 the process of maturation is seen to be com- 
pleted, the polar globules lying in a slight depression on the dorsal surface 
of the egg. The 2-cell stage is shown in Figure 20. The 4-cell stage is 
seen in Figure 21 to be approaching, and has been reached at the stage 
shown in Figure 22. The two blastomeres on the side toward the 
observer appear to be of equal size, the other two are hid from sight. 
A view of the egg immediately after the next division is shown in Figure 
23; the appearance nine minutes later is shown in Figure 24. These 
both represent the 8-cell stage, and show that the four cells which lie 
nearest the polar globules are smaller than those more remote. They also 
show that division has occurred in such a manner that the pair of cells 
occupying the upper right-hand corner of the figure is in contact with 
the diagonally opposite pair of cells in the lower left-hand corner of the 
figure, whereas the pair of cells in the upper left-hand corner is entirely 
separated from that diagonally opposite it. This arrangement is due to 
no accidental shoving of cells one over another, but is found invariably 
occurring at the 8-cell stage. The diagonally opposite cells which are in 
contact form respectively the posterior dorsal and anterior ventral por- 
tions of the embryo. This arrangement of the cells of the 8-cell stage 
has up to the present time been overlooked by all writers on tunicate 
embryology except Chabry (87). He both distinctly recognized and 
clearly figured it. (See his Planche XVIII. Fig. 9.) But, as I pointed out 
in a previous paper (’94), that hemisphere of the egg which he, following 
Van Beneden et Julin, called dorsal, was really the ventral hemisphere, 
so that he wrongly calls the cells in contact the anterior dorsal and pos- 
terior ventral. If we correct his naming of the hemispheres, his obser- 
vations on Ascidiella are brought into complete agreement with mine on 
Ciona regarding this point. In both cases the posterior dorsal and ante- 
rior ventral cells of the 8-cell stage are in contact. Though Seeliger (’85) 
apparently overlooked the fact, his figures (Taf. I. Figs. 7, 8, and 10), 
when their orientation is corrected as I (94) have shown to be necessary 
for other reasons, present precisely the same arrangement of cells in the 
8-cell stage of Clavelina. This condition is therefore probably of general 
occurrence among the simple and social Ascidians. 

The 16-cell stage immediately after its formation is shown in Plate IV. 
Fig. 25, and half an hour later in Figure 26. In the stage represented 
by Figure 26, spindles, directed as indicated by the arrows, were already 
visible in the large cells, occupying the lower half of the figure, though 
none had yet appeared in the smaller cells composing the upper half of 
the figure. This fact foreshadows an earlier division on the part of the 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 229 


cells of the lower hemisphere, which would lead to a stage of twenty-four 
cells. Such a stage was figured in my preliminary paper (’94, Plate I. 
Figs. 1 and 2; here reproduced in Plate IX. Figs. 51 and 52), and it 
was there demonstrated that the hemisphere in which division is earliest, 
as the egg passes from the 16-cell stage, becomes later the ventral or 
ectodermal hemisphere of the embryo. 

Accordingly the series of observations illustrated by Figures 19-26 
goes to prove that the four larger cells of the 8-cell stage, which are 
more remote from the polar globules, form the ventral or ectodermal 
half of the embryo, whereas the four smaller cells, on which the polar 
globules rest, become the dorsal or endodermal half of the embryo. 

The same thing is shown by Figures 27—34 (Plates V. and VI.), a series 
of drawings of an egg viewed from its anterior end. In Figures 27-29 are 
seen successive phases of the 2-cell stage. Figure 30 shows the 4-cell 
stage, and Figures 31 and 32 two phases of the 8-cell stage. At the 
8-cell stage in this series, as well as in the series previously examined, 
the four cells nearest the polar globules are smaller than the other four ; 
they will form, as we shall see, the dorsal hemisphere. There has been 
no shoving of cells across the median plane, but shoving has occurred 
among the cells of the right and left halves of the embryo separately, as 
was seen also at this stage in the series previously examined. (See Plate 
IV. Fig. 23.) According to the rule already stated, we should find in 
contact with each other the diagonally opposite pairs of cells which are 
to form respectively the posterior dorsal and anterior ventral portions of 
the embryo; while the other two pairs of cells should be completely 
separated. If this is true in the case before us (Figs. 31 and 32), we 
are looking at the anterior end of the embryo, for the pair of ventral cells 
nearest the observer is seen to be in contact with the most remote pair 
of dorsal cells. 

Figure 33 (Plate VI.) shows the 16-cell stage, and Figure 34 the 
24-cell stage in process of formation. In this egg also the cells of 
the hemisphere most remote from the polar globules were first to divide 
in passing from the 16-cell stage. Those of the other hemisphere divided 
in this case about twelve minutes later. Therefore by this series also the 
hemisphere more remote from the polar globules is shown to be the ventral 
or ectodermal. That one is looking in this series at the anterior end of 
the embryo, as already suggested, and not at the posterior end, is shown 
by a comparison of Figure 34 (Plate VI.) with Figure 51 (Plate IX.), 
both of which represent the 24-cell stage. The posterior end of the em- 
bryo is seen in Figure 51 to be marked by a noticeably small pair of 


230 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


cells, the like of which does not appear in Figure 34, but may be sup- 
posed to he hidden from view at the more remote end of the embryo. 
Moreover, the cells A%1, 4&?, B%1, 5%? of Figure 51, which are situated 
at the anterior end of the embryo, correspond well in size with the four 
cells nearest the observer in Figure 34. Therefore the rule previously 
stated for the orientation of the 8-cell stage is exemplified in this series 
also. 

In Figures 45-50 (Plate VIII.) is shown another series of drawings 
illustrating what has been said regarding the clear protoplasmic region (2) 
which throughout cleavage marks the posterior end of the embryo. In 
this series one looks down obliquely on the dorsal surface of the em- 
bryo from its posterior end. The polar globules are not visible, for the 
reason that they do not come into profile at the margin of the egg, 
a circumstance which is necessary for an exact determination of their 
position. 

In Figure 45,a 2-cell stage, the clear region appears in each blastomere 
at x. During each successive cell division it bulges out as represented 
in Figure 46, and again in Figure 47, just as if it were the most plastic 
portion of the egg and responded most readily to the internal tension 
which accompanies cell division. Such indeed is probably the case, for 
this region is free from yolk granules, consisting of protoplasm only, as 
has been already pointed out. 

In Figure 48, the 8-cell stage is seen to be completely formed. 
Applying our rule for the orientation of the egg at this stage, we decide 
that the pair of cells occupying the centre of the figure and nearest to 
the observer is to form the posterior dorsal portion of the embryo ; 
for (1) it belongs to the set of four smaller cells formed by the first 
equatorial plane of cleavage, and (2) it is in contact with the diagonally 
opposite pair of cells of the other hemisphere. The sequel justifies our 
conclusion. Figure 49 represents the 16-cell stage, and Figure 50 
the 24-cell stage.t In Figure 50 it is seen that the small posterior cells 
of the ectodermal hemisphere, unmistakably identical with O*%? and D®® 
of Figure 51 (Plate IX.), have appeared just where the clear portions 
forming prominences at the time of cell division have all the time been. 
These portions have become a part of the small cells in question, which 
contain less yolk than any other cells of the egg at this stage, and 
subsequently cleave less rapidly than any other cells of the ventral 

1 Tt will be observed that between the stages represented in Figures 49 and 50 


there has been a slight rotation of the egg, so that the latter figure exhibits an 
exactly dorsal view instead of an obliquely dorsal one. 


9 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 231 


hemisphere. The persistence of this clear polar region in stages later 
than that of 24 cells was shown in certain figures of my preliminary 
paper (reproduced in Plate IX. Figs. 54 and 55). It finally passes into 
the small flattened cells C7, D'® (Plate XI. Fig. 71), of whose later 
history we shall have more to say. 

Chabry (?87) observed in Ascidiella at the beginning of the 8-cell 
stage the formation of polar prominences such as I have described, 
and spoke of them as a sure means of orienting the egg at this stage. 
On page 203 he says: “Il est encore une marque propre aux cellules 
P et P [.D*1, C**] que permet de les distinguer de toutes les autres, elle 
consiste en une petite saillie en forme de mamelon, saillie qui est dirigée 
horizontalement en arriére et que montrent les figures 2 et 23 de la 
planche XVIII. Cette saillie n’est visible qu’au début du stade VIII. 
[8-cell] et surtout durant la segmentation qui produit P et P [D*,0*1].” 
Apparently Chabry overlooked the formation of the prominences at 
other than the 4- and 8-cell stages, and failed to recognize their true 
significance. For he explains them as merely foreshadowing the form 
and direction of the next cell division, and as referable to a supposed 
general phenomenon, which, stated in his own words, is as follows: “ Que 
les blastoméres ont 4 l’instant ot ils viennent de se produire et mieux 
encore durant leur individualisation des formes spécifiques quils perdent 
peu d'instants aprés. Ces formes spécifiques paraissent étre en rapport 
avec les segmentations dont ces blastoméres seront plus tard le siége .. . 
la segmentation a done lieu dans tous les cas, perpendiculairement au 
plus grand axe que possedait, le blastomére durant son individualisation.” 

It is hardly necessary, I suppose, to say anything at this late day in 
refutation of Chabry’s generalization. My own observations indicate 
that cells tend to assume at the time of their formation (“ individualisa- 
tion”) a spherical form,’ if they are homogeneous in structure, and that 
the departure from an evenly rounded contour at the posterior end of the 
ventral hemisphere is explicable by the presence there of a region pecu- 
liar in its constitution, containing as it does less yolk than the other 
superficial portions of the egg.” 


1 Mutual pressure of cells may modify this form, in which case the direction of 
the next division may perhaps be predicted, as Chabry states, at the time of the 
“individualisation ” of cells. For, other things being equal, it is true that the spindle 
arises in the longest axis of the cell. 

2 IT am aware that Van Beneden et Julin (’84) have offered an entirely differ- 
ent explanation for certain phenomena probably related to those under discussion, 
which they observed in the cleaving egg of Clavelina. Their explanation implies 


232 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


Let us examine still another series of drawings (Figs. 35-42, Plates 
VI. and VII.) made from the living egg, which in this case is viewed _ 
from the ventral side and a little obliquely. The polar globules of — 
course are not seen, since they lie on the opposite side of the egg. — 
Neither is the point of view a favorable one to bring the posterior polar 
regions clearly into profile as in the series last examined. 

Figure 35 (Plate VI.) shows the 4-cell stage; Figures 36 and 37, 
successive views of the 8-cell stage; and Figure 38, a 12-cell stage, the 
four cells of the ventral hemisphere having divided in this case a little 
earlier than those of the dorsal hemisphere. This is unusual, for the 
difference in rate of cleavage of the cells of the two hemispheres com- 
monly first appears, as we have seen in the three series previously 
examined, in passing from the 16-cell stage to one of 24 cells. 

Figure 39 (Plate VII.) gives a view of the egg five minutes after the 
stage shown in Figure 38 had been reached. It represents the 16-cell 
stage. A drawing made five minutes later still is shown in Figure 40, 
and one made ten minutes after that is shown in Figure 41. 

In the last mentioned figure, the cells of this uppermost hemisphere 
are seen to have again become rounded in outline preparatory to the 
next cell division. Spindles are already visible in them, as indicated by 
the arrows, those last to appear being the ones in the small cells 
(C*?, D®*) at the lower margin of the figure. The subsequent division 
was about a minute later in these two cells than in the others of the 
same hemisphere ; this is regularly the case in the cell division which 
leads to the 24-cell stage. 

Figure 42, the last of the series, will be at once recognized, by one 
who has read my preliminary paper, as a ventral view of the 24-cell 
stage. (Cf. Plate IX. Fig.51.) The posterior end is clearly marked by 
the small cells 0%, D®*. A re-examination of Figures 36 and 37 
(Plate VI.) shows that the rule previously stated for orienting the egg 
at the 8-cell stage is again exemplified in the case of this series, for in 


the existence during karyokinesis of astral fibres which attach to the cell wall at 
particular points and by their contraction depress its surface. 

Such an explanation seems to me inadequate, at least for this case; first, because 
I have seen no evidence of the existence of astral fibres in karyokinesis ; secondly, 
because at successive cleavages the prominences appear in the same structurally 
peculiar region, whether the karyokinetic spindle is directed toward that region — 
as the explanation of Van Beneden et Julin would imply — or not (see Plate VIII. 
Fig. 47) ; thirdly, because astral fibres, if present, should appear in every blastomere 
at karyokinesis, but I have been able to discover these peculiar prominences only 
in the particular regions already described. 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 233 


Figures 36 and 37 we see in contact cells which we know, from an ex- 
amination of Figure 42, eventually become the anterior ventral and 
posterior dorsal portions of the embryo. 

We have now followed the cleavage cell by cell to the 24-cell stage. 
We have seen that cleavage is from the very beginning bilateral, and 
progresses in a very definite manner and at a very definite rate. This 
we shall find is true in the further development of the egg, even until 
the complete closure of the blastopore. Wilson (’94) observed that the 
cleavage of Amphioxus showed all gradations between a perfectly radial, 
a bilateral, and even a spiral form; and he raised a query whether the 
same might not be found to be true for Ascidians. In Ciona at least this 
does not seem to be the case. I have never observed an instance of 
deviation from the regular mode of cleavage described in the foregoing 
paper, unless one so construes the occasional very slight difference in the 
time of cleavage of the cells of the two hemispheres in passing from the 
8-cell stage, a matter to which allusion was made on page 232. No 
rotation of the cells of one hemisphere over those of the other even in 
the slightest degree bas ever been observed. In having a perfectly 
definite and stereotyped manner of cleavage, the ascidian egg resembles 
more closely the egg of Annelids, Mollusks, and the great majority of 
Invertebrates, than it does that of Amphioxus and the Vertebrates, not- 
withstanding that the end product of cleavage shows unmistakably the 
now generally admitted closer affinity of Tunicates with the latter group 
of animals. 


It remains to call attention to some of the internal phenomena accom- 
panying the early cleavage stages. The first cleavage spindle arises, as 
has been stated, not far from the centre of the egg. (See Plate III. 
Fig. 14.) As its first cleavage is nearing completion, however, the 
attraction spheres and nuclei begin to move toward the dorsal surface o 
the egg, away from its more richly protoplasmic (animal) pole, from 
which the plane of separation cuts in more rapidly. (See Plate ITI. 
Fig. 15.) The attraction sphere of each blastomere grows more diffuse 
as the nuclei pass into a resting condition; it then elongates in a hori- 
zontal direction and parallel to the first plane of cleavage, and finally 
divides. The parts separate and the nucleus moves out to a position be- 
tween them. (See Plate III. Fig. 16.) By this time the attraction 
spheres and nuclei unmistakably lie closer to the dorsal (maturation) 
surface of the egg. (Plate III. Fig. 16; cf. Plate IV. Figs. 20, 21, and 
Plate V. Figs. 27-29; also Van Beneden et Julin’s [’84] Figs. 2 and 46, 


Ol, OOUE_—=InGh YE 3 


234 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


Plauche VII., remembering that dorsal and ventral are reversed in Van 
Beneden et Julin’s figures.) The yolk-free protoplasm trails downward 
from the attraction spheres forming a sort of crescent in each blasto- 
mere. (See Plate III. Fig. 16.) 

During the second and third cleavages the nuclei remain somewhat 
nearer the dorsal (vegetative) pole. (Plate IV. Figs. 20-22, and Plate V. 
Fig. 30; cf. Van Beneden et Julin’s [’84] Figs. 4 6 and 5, Planche VII.) 
It follows naturally that when the 8-cell stage is formed by the first 
equatorial plane of cleavage (third cleavage), an inequality is observed 
in the size of the newly formed blastomeres, the four nearer to the dorsal 
pole being smaller than their sister cells, though the latter are richer in 
protoplasm. (See Plate IV. Fig. 23, and Plate III. Fig. 18.) 


(6) Summary on Early Cleavage Stages. 


1. The future posterior end of the embryo is marked at the 2-cell 
stage by an accumulation of protoplasm free from yolk in each blas- 
tomere at contiguous regions. This accumulation persists throughout 
cleavage, and forms at each cell division a pair of protuberances beyond 
the general contour of the blastomeres. 

Subsequently to the 8-cell stage, in each of the two blastomeres in 
which these accumulations lie, the spindle at three successive cell 
divisions is directed toward the protoplasmic accumulation of that cell 
and lies nearer to it than to the opposite side of the cell. In conse- 
quence the newly formed cell, which contains the region in question, is 
in each case smaller than its sister cell. (Cf. D®* and D*?, Fig. 38, 
Plate VI. ; .D*? and D®**, Fig. 51, Plate IX.; and D’* and D"', Fig. 62, 
Plate X.) 

At each of these divisions also cleavage occurs Jater in the cells con- 
taining the protoplasmic accumulations than in their sister cells. 

2. The first cleavage plane is vertical, and passes through the point of 
formation of the polar globules. It coincides with the future median 
plane of the embryo, and divides the egg into two blastomeres equal in 
size and similar in every particular. They form respectively the right 
and left halves of the embryo. The fate, as just stated, of the first two 
blastomeres of the ascidian egg was first pointed out in the case of 
Clavelina by Van Beneden et Julin (’84). 

3. The second cleavage plane is also vertical, and at right angles to 
the first. Like the first, it passes through the point of formation of the 
polar globules. It divides the egg into four blastomeres, among which 
no difference of size can be recognized. 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 235 


4, The third cleavage plane is at right angles to both the preceding, 
i. e. equatorial in position. It separates four smaller cells lying nearer 
to the polar globules and more abundantly supplied with yolk, from four 
larger ones more remote from the polar globules and richer in protoplasm. 

The former are destined to give rise to the dorsal or endodermal 
hemisphere of the embryo; the latter, to the ventral or ectodermal 
hemisphere. 

The protoplasmic accumulations mentioned under paragragh 1 always 
fall in the posterior pair of cells of the ventral hemisphere (viz. D*+, 041) 
close to its line of contact with the dorsal hemisphere. This pair of cells 
is never in contact with the anterior pair of cells of the dorsal hemi- 
sphere, but the anterior pair of cells of the ventral hemisphere (viz. 
A‘, B*) is invariably in contact with the posterior pair of cells of the 
dorsal hemisphere (viz. c*?, d*). 

The fact just stated affords a ready and unfailing means of orienting 
the 8-cell stage. This was recognized by Chabry in the case of Asci- 
diella, and is shown by an examination of Seeliger’s figures to be equally 
true for Clavelina. It probably holds good among all the simple and 
social Ascidians. 

5. The 16-cell stage is usually reached by simultaneous divisions in 
both hemispheres. Sometimes, however, the cells of the ventral hemi- 
sphere at this cleavage divide sooner than those of the dorsal hemi- 
sphere, thus giving rise to a 12-cell stage, but this very soon changes 
to a 16-cell stage by the cleavage of the cells of the dorsal hemisphere. 

6. As the egg passes from. the 16-cell stage, cleavage invariably occurs 
earlier in the cells of the ventral hemisphere, i.e. the descendants of the 
four larger cells of the 8-cell stage, than it does in the cells of the dorsal 
hemisphere. A 24-cell stage results, in which the cells of the ventral 
hemisphere, being twice as numerous as those of the dorsal hemisphere, 
cover more surface and begin the process of overgrowth (epiboly), for- 
cing the cells of the dorsal hemisphere into a somewhat columnar form. 
(See Plate VII. Fig. 44.) 


B. Later Staces or CLEAVAGE. 
(a) From 24-cell to 46-cell Stage. 


The 24-cell stage was taken as the point of departure in my prelimi- 
nary paper (’94), and the cell lineage was traced in detail through a 
stage of 46 cells. I shall not repeat except in the form of a brief résumé 
what was there said regarding those stages, but shall content myself 


bo 


36 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


with reproducing (in Plate IX.) the figures of Plate I. accompanying 
that paper, which were executed to illustrate this period of the develop- 
mental history. These figures present dorsal and ventral views of the 
24-cell stage (Plate IX. Figs. 51 and 52), the 32-cell stage (Figs. 53 
and 54), and the 46-cell stage (Figs. 55 and 56). 

The interpretation, as given in my preliminary paper, of the lineage 
through the 46-cell stage rested upon the strongest possible evidence, 
viz. the observation of karyokinetic figures for every cell division which 
was represented as having occurred. Moreover, it was shown that these 
observations made it possible to reconcile the conflicting statements of 
others who had studied the cleavage of the ascidian egg. Such excellent 
observers as Van Beneden et Julin, on the one hand, and Seeliger, on the 
other, held contrary opinions as to which was the dorsal side and which 
the anterior end of the embryo in its early stages in one and the same 
genus, Clavelina. 

It was shown in my paper, both from an examination of the authors’ 
own figures and from a comparison with the lineage of Ciona, that their 
conflicting statements arose from a fundamental error on the part of 
each, Van Beneden et Julin being correct in their determination of the 
ends of the embryo, and Seeliger in his determination of the dorsal and 
ventral surfaces of the early stages. Upon correcting these mistakes, it 
was found that the observations of the writers mentioned were brought 
into harmony, and were then in agreement with my own observations 
on Ciona. 

In order to demonstrate that I had correctly determined the dorsal and 
ventral faces of the egg for the 46-cell and earlier stages, in contradiction 
to the interpretation of Van Beneden et Julin, I figured a single older 
stage described as one of 66 cells (Castle 794, Plate II. Figs. 11 and 12). 
Its presentation was intended to bridge the gap between the 46-cell stage 
and gastrulation. This purpose it fulfilled, for it showed gastrulation 
already commenced, and so proved beyond question which was to be the 
oral (dorsal) and which the aboral (ventral) surface. 

A desire to give completeness to my figures led me to state the lineage 
of this stage as I then understood it. I have since found, from the study 
of more complete series of embryos than I had at that time secured, 
that I was mistaken as to the time of cell division in one pair of cells 
(078, DV’, Fig. 56, Plate IX.). I supposed it had already occurred 
at the stage represented in Figures 11 and 12 (Plate II.) of my former 
paper. Consequently the lineage there given for this stage is incorrect. 
Though this fact does not affect the main conclusions of my preliminary 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 237 


paper, it necessitates modification of several minor statements, as will be 
indicated in detail later. 

The 24-cell stage, it has been seen, arises from the 16-cell stage by an 
earlier division on the part of the cells of the ventral hemisphere than 
occurs in those of the dorsal hemisphere. Accordingly, we find that at 
the 24-cell stage the ventral hemisphere consists of sixteen cells, whereas 
the dorsal hemisphere is made up of only eight. These eight are com- 
pressed into a columnar form by the overgrowth of the cells of the 
ventral hemisphere already begun. (See Plate VII. Fig. 44.) Their nu- 
clei lie in a superficial position, while their deep ends are heavily laden 
with unassimilated yolk. They retain this columnar form up to and 
throughout gastrulation. In number, they are soon brought up to an 
equality with the cells of the ventral hemisphere by division, which 
leads to the 32-cell stage (Plate IX. Figs. 53 and 54) and places all the 
cells of the egg in the sixth generation. 

Presently the cells of the ventral hemisphere again anticipate in 
division those of the dorsal hemisphere, this time by a still longer inter- 
val. Among the cells of the ventral hemisphere differences in the time 
of division could, as we have seen, be detected at the preceding cleavage. 
At the present cleavage the differences become more pronounced. In 
particular, the small posterior cells, C®°, D°* (Plate IX. Figs. 53 and 54), 
divide enough later than their fellows to allow us to recognize a 46-cell 
stage (Plate IX. Figs. 55 and 56), made up as follows : — 


Ventral hemisphere, 28 cells in the seventh generation, 
2 cells (C'%?, D®?) in the sixth generation. 
Dorsal hemisphere, 16 cells in the sixth generation. 


46 
When the two small cells C®*, D®*, divide, which they do earlier than 
the cells of the dorsal hemisphere, a stage of forty-eight cells is reached, 
all the cells of the ventral hemisphere (thirty-two in number) being in 
the seventh generation, and those of the dorsal hemisphere (sixteen in 


number) being in the sixth generation. Such a stage is shown in Plate X. 
Figs. 57 and 58. 


(6) 48-cell Stage. 


The embryo shown in Figures 57 and 58 has a vertical axis the length 
of which is equal to that of its antero-posterior axis, if not greater. 
Accordingly it has been found easier to maintain this axis in a horizon- 
tal position, and hence more convenient to represent the egg as viewed 


238 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


from the anterior and posterior ends respectively, rather than from the 
dorsal and ventral surfaces, as in most of the other stages figured. This 
stage (48-cell) is made up as follows : — 


Ventral hemisphere, 32 cells in the seventh generation. 
Dorsal hemisphere, 16 cells in the sixth generation. 


48 


It will be observed that the cells of the ventral hemisphere, though all 
in the seventh generation, are not all equally advanced in their prepara- 
tions for division, which evidently is again about to set in. For while 
the cells occupying the centre of the ventral hemisphere, or, in other 
words, lying nearest to the animal pole of the egg, are about to pass 
into the next generation, the cells occupying the margin of the ventral 
hemisphere, and more remote from the animal pole, contain nuclei en- 
tirely quiescent, like those seen in the cells of the dorsal hemisphere. 
This is contrary to the statement made in my preliminary notice (’94), 
in which I said that at this division those cells of the ectodermal hemi- 
sphere which were marginal and in contact with cells of the endodermal 
hemisphere were first to divide. This erroneous statement arose from 
the wrong interpretation given to Figures 11 and 12 (94 Plate II.) in 
describing the cell lineage of that stage, a matter to which attention has 
already been directed. 

In the embryo shown in Plate X. Figs. 57 and 58 (48-cell stage) one 
may readily distinguish three regions, each composed of sixteen cells. 
The first region is the dorsal hemisphere, with its sixteen cells all in the 
sixth generation (a®>-a**, d°5-d®*, and the corresponding cells in quad- 
rants Band (). These cells are destined to form the endoderm of the 
larva, the chorda, and a portion of the mesoderm. The second group of 
sixteen cells occupies the centre of the ventral hemisphere (A™, A™?, A™3, 
Al}, Atl, Di, D7, and D™3, with the corresponding cells in quadrants 
Band C). They are in the seventh generation, but already contain 
spindles, showing that they are soon to pass into the eighth generation. 
This group of cells will form the ectoderm of the larva. The remain- 
ing sixteen cells of this embryo, also belonging to the ventral hemi- 
sphere, form the third group (A™4, A™*, AT’, and D'™*—D"™, with the 
corresponding cells in quadrants Band ©). They too are in the seventh 
generation, but their nuclei are quiescent, showing that these cells will 
be later in dividing than the other cells of the ventral hemisphere. 
They are arranged in an equatorial band between the other two groups 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 239 


of cells. This band is interrupted at only one point on each side of the 
embryo, where a single cell (47°, Bt°) of the ectodermal group reaches 
up into contact with the cells of the dorsal hemisphere. From the equa- 
torial band just described are derived chiefly nerve cells and mesoderm 
cells. 


(ce) G4-cell Stage. 


The completion of the divisions foreshadowed by spindles in the ecto- 
dermal group of cells of the stage last discussed (Plate X. Figs. 57 

and 58) doubles the number of cells in that group, and brings the num- 
_ ber in the entire embryo up to sixty-four, distributed as follows. 


Ventral hemisphere (designated by the letters A, B, C, D) :— 
32 cells in the 8th generation =the ectodermal group. 
16 7th i =the equatorial band. 


48 
Dorsal hemisphere (designated by a, 6, ec, d) :— 
16 cells in the 6th generation. 


64 


Such a stage is shown in Plate X. Figs. 59 and 60, the former 
representing a ventral and the latter a dorsal view. The egg has again 
assumed the flattened form which it had at the 32-cell stage. 

Examining first the ventral surface (Fig. 59), we see that the divisions 
foreshadowed in the 48-cell-stage (Figs. 57 and 58) have in every in- 
stance occnrred in a direction perpendicular to that of the spindle in 
the mother cell, though a slight displacement is in some cases appear- 
ing among the daughter cells, on account of the mitoses arising in the 
equatorial band. The cells of the ectodermal group, on account of their 
recent division, now number thirty-two, as many as are found in both 
the other groups put together. They are in the eighth generation, one 
generation in advance of the cells of the equatorial band, and two gen- 
erations in advance of the cells of the dorsal hemisphere. They are 
A®1_ ASS, 489, 4810 4818 4514 and D®!—D%* together with the correspond- 
ing cells in quadrants Band C. 

The equatorial band is, as at the last stage, composed of sixteen cells 
all in the seventh generation, but six of them (three on each side of the 
median plane, Fig. 60, A™4, A™’, and D'*) now show signs of approaching 
division. Four of these mitotic cells form the anterior segment of the 
equatorial band, and are destined to produce a considerable portion of 


240 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


the nervous system of the larva. (See Fig. 60, A™*, A™8, B”4, and B™*) 
The two remaining mitotic cells of the equatorial band are situated later- 
ally one each of the posterior quadrants (Figs. 59 and 60, D'4, 07), 

The ten remaining cells of the equatorial band all contain resting 
nuclei. Eight of these cells are grouped at the extreme posterior end of 
the equatorial band in a region where, from the 16-cell stage on, we have 
found cleavage to be more tardy than in any other part of the ventral 
hemisphere. These eight cells are D™*, D'°, D7, D™§, and the corre- 
sponding cells in quadrant C. (Figs. 59 and 60. Compare Fig. 57.) The 
two remaining cells of the equatorial band which still show no signs of 
division are A™® and 5’-® (Fig. 60), situated about midway between the 
anterior and posterior ends of the embryo. 

Of the sixteen cells comprising the dorsal hemisphere (Fig. 60), six, 
which lie in contact with the equatorial band (a®*, a®’, d®*, with their 
mates in quadrants & and C), are in mitosis. Four of them, the most 
anterior of the cells of the dorsal hemisphere, lie in a transverse row 
across the dorsal surface of the embryo (Fig. 60, a*7, a&*, 6%, and 6°"). 
They will ultimately form the greater portion of the chorda. We will 
call them the antertor chorda fundament. The two other mitotic cells 
of the dorsal hemisphere are d®* and e¢®*, in the posterior half of the 
embryo (Fig. 60). The spindles in these cells are directed obliquely 
forward, upward, and outward, so that, taking into consideration the 
superficial position of the nuclei of the dorsal hemisphere, we may pre- 
dict that the coming division will result in cutting off in each case a 
smaller more superficial cell from a larger cell extending deeper; the 
small cell will also lie anterior and lateral to its sister cell. The unequal 
divisions in these two cases will separate cells of unlike fate; the two 
smaller cells will constitute the posterior chorda fundament, the two 
larger ones will form mesoderm. 

The ten remaining cells of the dorsal hemisphere (Fig. 60, a®*, a®%, 
d®*, d®7, d°8, and the corresponding cells in quadrants B and C’) show as 
yet no signs of division. They are grouped about the vegetative pole of 
the egg, the point of origin of the polar cells, and will form the whole 
of the definitive endoderm of the larva, and nothing else. 


(d) 76-cell Stage. 


Upon the completion of division in the twelve mitotic cells of the 
embryo represented in Plate X. Figs. 59 and 60, a stage of seventy-six 
cells would be reached. An embryo in this stage is shown in Plate X. 
Figs. 61 and 62. It contains in the 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 241 


Ventral hemisphere (designated by the letters A, B, C, D): 
32 cells in the eighth generation = the ectodermal group. 
12 cellsinthe “ 2 
10 cells in the seventh ‘“ 

54— 
Dorsal hemisphere (designated by a, 6, e, d): — 
2 mesoderm cells in the seventh generation. 

10 chorda cells in the bs ie 
10 endoderm cells in the sixth Be 


= the equatorial band. 


S| 
oO! b 


The ectodermal group of the ventral hemisphere contains the same 
number of cells as at the 64-cell stage, viz. thirty-two, —sixteen on 
each side of the median plane, ten of them being derived from an ante- 
rior quadrant (A), six from a posterior quadrant (D). They cover 
nearly the entire ventral surface of the egg. (See Plate X. Fig. 61, 
ae ASS ARP APO Ae18 Aei4 and .D*—D** as well as the corre- 
sponding cells in the right half of the figure.) All the cells of this group 
are in the eighth generation. 

The equatorial band now contains six more cells than at the 64-cell 
stage, in consequence of the completion of divisions foreshadowed at 
that stage in the cells A’-*, A’:’, B'* B®, C74 and D'* (Fig. 60). 
It now consists of twenty-two cells, which, in passing from the posterior 
end forward, are D™-5, Di-6 Di-7, Di-8, D8-7, D8-8, Av, 48-16 48-15 A883 
and A®-7, with the corresponding cells in quadrants B and C (Fig. 62). 
Six of the cells on each side of the median plane are derived from a 
posterior and five from an anterior quadrant. Signs of approaching di- 
vision have at this stage become visible in four of the cells of this equa- 
torial band, viz. A™-®, B’-®, D7-7, and O7:7, In the case of the first two 
cells mentioned the spindles stand vertically (cf. Plate X. Fig. 67, A™-) ; 
in the other two cells (C7", D’-") the spindles are nearly horizontal in 
position, though their antero-lateral ends lie at a slightly higher level 
than the opposite ends. 

There are only six cells remaining in the equatorial band which nei- 
ther have passed into the eighth generation nor show any signs of imme- 
diately doing so. They are grouped at the posterior end of the embryo, 
which has been repeatedly pointed out as the region of slowest cleavage 
among the cells of the ventral hemisphere. The six cells in question 
are D™*, D™°, Di’, and the corresponding cells in quadrant C (Fig. 62). 


242 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


Although for convenience I shall continue to use the term equatorial 
band, it is clear that the cells composing it are no longer strictly equato- 
rial in position, but now lie on the flattened dorsal surface (Fig. 62). 
This change of position has come about in consequence of the more 
rapid cell division in the ventral hemisphere. How considerable the dif- 
ference in rate of division has been between the cells of the two bemi- 
spheres, one readily appreciates if he stops to consider that the cells of 
the ventral hemisphere now number fifty-four, whereas those of the 
dorsal hemisphere number only twenty-two. 

In the dorsal hemisphere (Fig. 62) the divisions foreshadowed by 
spindles at the 64-cell stage (Fig. 60) have taken place, but no new 
ones are approaching. The number of cells in this hemisphere is now 
twenty-two; twelve of them (the chorda and mesoderm cells, ef. descrip- 
tion of Fig. 62 in the explanation of Plate X.) are in the seventh gen- 
eration, and ten (the endoderm fundament), in the sixth generation, no 
divisions having occurred in the last named group of cells since the 32- 
cell stage. Of the ten chorda cells, eight derived from the anterior quad- 
rants are arranged in a crescent-shaped band capping the anterior end of 
the dorsal hemisphere ; they are a™°, a, a™8, a™-"4, and the correspond- 
ing cells in quadrant 6. They form the anterior chorda fundament. 
The other two chorda cells, which are derived from the posterior quad- 
rants, are d™" and c™. They form the posterior chorda fundament, 
and are at present separated from the anterior chorda cells by two cells 
of the equatorial band, viz. A™* and B'*. 

The sister cells of d7-" and ec’), viz. d7: and c™-?, are the sole con- 
tribution of the dorsal hemisphere to the mesoderm of the larva, for the 
greater part of the mesoderm is, as we shall see, derived from the equa- 
torial band. 

Among the endoderm cells it is noticeable that d®* and its mate e®* 
have been shoved forward out of their own quadrants to a position 
beside the endoderm cells derived from the anterior quadrants. 


(e) Summary on Later Cleavage Stages. 


1. In the cleaving ovum one can recognize, in passing from the ani- 
mal to the vegetative pole successive zones, in each of which cleavage 
takes place less rapidly than in the preceding. At the 64-cell stage 
(Plate X. Figs. 59, 60) there are three such zones: first, a group of 
thirty-two cells encircling the animal pole, all of them in the eighth 
generation ; second, an equatorial zone of sixteen cells, all in the seventh 
generation ; third, a group of sixteen cells encircling the vegetative pole, — 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 243 


all in the s¢xth generation. ‘The first two zones are descended from the 
four ventral cells of the 8-cell stage, i. e. from the four cells most remote 
from the point of formation of the polar globules. The third zone is 
descended from the four dorsal cells of the 8-cell stage. The ectoderm 
is derived chiefly from the first zone, — that is, the zone encircling the 
animal pole ;—the mesoderm is derived chiefly from the second zone, 
and the endoderm exclusively from the third zone. 

This zonal arrangement persists throughout cleavage and the early 
stages of gastrulation, but its symmetry is at each succeeding stage dis- 
turbed to an increasing extent by the fact that cell division is less rapid 
at the posterior than at the anterior end of the embryo. 

2. Although, as just stated, cleavage progresses with unequal rapidity 
at the two poles of the antero-posterior axis, as well as at those of the 
dorso-ventral axis, it is equal in rate at the two poles of the third axis 
of the egg, viz. the transverse. The last mentioned fact serves to main- 
tain the perfectly bilateral form of the embryo. 

The differentiation of the poles of the dorso-ventral and antero-poste- 
rior axes, the reader will recall, was already recognizable by structural 
cytoplasmic differences in the unsegmented ovum. Zhe form and rate of 
cleavage are therefore manifestly predetermined by the internal constitution 
of the ovum. 


3. Gastrulation. 
A. Earty SraGes or GASTRULATION. 


(a) 112-cell Stage. 


An embryo a little more advanced in development than the one 
represented in Figures 61 and 62 (Plate X.) is shown in dorsal view 
in Figure 71 (Plate XI.). No new divisions have occurred in the 
dorsal hemisphere, which accordingly consists, as at the last stage, of 
twenty-two cells. In the equatorial band, the four cells which were 
preparing for division at the 76-cell stage (Plate X. Fig. 62, D™:7, C77, 
A’™®, and B"*) are seen in Figure 71 to have divided, though in the case 
of A™-° and 5°, on account of the vertical position of the spindles (ef. 
Fig. 67), only the more superficial daughter cell is in each instance visi- 
ble (A’-?, B82, Fig. 71). No further divisions have occurred in the 
equatorial band, which therefore consists at this stage of twenty-six cells, 
all in the eighth generation except the group of six cells arranged in 
crescent form at the posterior end of the embryo, viz. D™*, D™°, D7’, and 
the corresponding cells in quadrant C. These cells have lingered in the 
seventh generation later than all other cells of the ventral hemisphere. 


244 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


The equatorial band as a whole has now moved to a position distinctly 
within the margin of the dorsal surface (Fig. 71), so that a row of cells 
from the ectodermal group of the ventral hemisphere has come into view 
outside it round almost the entire periphery of the embryo (ef. Fig. 62). 
This change has come about in consequence of additional divisions in the 
ectodermal group of cells, which now not only has spread over the entire 
ventral surface of the embryo, but is encroaching upon its dorsal surface. 

Division has occurred nearly synchronously in all the cells of the ecto- 
dermal group, though somewhat sooner in those nearest the animal pole. 
(See Plate X. Figs. 63-70.) The strongest possible confirmation of my 
own observation regarding the simultaneousness of division in the cells 
of the ectodermal group in this period of development is afforded by 
Samassa’s (’94) Figures 10 and 11, Taf. Il. These represent respectively 
a dorsal and a lateral view of a stage intermediate between those shown in 
my Figures 62 and 71. In Samassa’s Figures 10 and 11, all the cells of 
what I have called the equatorial band are figured as containing qui- 
escent nuclei, except the four seen to be mitotic in my Figure 62 
(Samassa’s cells 3 and 6, Fig. 10). The other cells of the ventral 
hemisphere visible in Samassa’s figures are without exception in process 
of division. It may accordingly be confidently assumed that at the stage 
shown in Figure 71 the cells of the ectodermal group of the ventral 
hemisphere have all passed into the ninth generation. If so, they number 
sixty-four ; this agrees well with the approximate count which one can 
make from dorsal and ventral views, though it is impossible to be sure 
about the exact number of ectoderm cells lying at this stage in an equa- 
torial position between the dorsal and ventral surfaces. Accordingly I 
shall not attempt to give for this and subsequent stages the lineage of 
the individual cells of the ectodermal group. This would be a work of 
great difficulty and of some uncertainty, for in this case the cells entirely 
lack those marked differences of size, stainability, and arrangement 
which make the lineage for the cells of the equatorial band and dorsal 
hemisphere a matter of perfect definiteness. 

If the estimate given of the number of cells in the ectodermal group _ 
is correct, the embryo shown in Plate XI. Fig. 71 represents a stage of 
one hundred and twelve cells distributed as follows. 

Ventral hemisphere : — 
64 cells in the 9th generation = the ectodermal group, 
20 - Sth as ) . 
6 ¥ wth = « 5 = the equatorial band. 
90: 


eee 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 245 


Dorsal hemisphere : — 
2 mesoderm cells in the 7th generation. 


10 chorda Ke 7th = 
10 endoderm zs 6th a 
22 — 
90 (in ventral hemisphere). 
112 


The process of gastrulation has at this stage already set in. Not only 
is the ectoderm growing over so as to envelop the dorsal hemisphere, 
but the latter is at the same time sinking down and becoming saucer- 
shaped. (Cf. Figs. 66 and 77.) Accordingly, gastrulation may be said 
to take place by a combination of the two processes of epiboly and 
invagination. 


(6) Differentiation of the Principal Organs as seen at the 112-cell 
Stage. 
a. TOPOGRAPHICAL. 

We will now consider this same embryo (Plate XI. Fig. 71) with 
reference to the ultimate fate of its cells. At the depressed centre of 
its dorsal surface, surrounding the point of formation of the polar glob- 
ules, we find the ten cells of the definitive endoderm, all in the sixth 
generation and containing each a very large nucleus. They are a®*, a®8, 
d®>,d°8, d®", and the corresponding cells in quadrants Band CO. Two 
of them are derived from each of the anterior quadrants (A and #), and 
three from each of the posterior quadrants (Cand D). Together they 
constitute the entire fundament of the definitive larval endoderm. 

The endoderm fundament is surrounded by two concentric rows of 
cells from which are derived some of the most important organs of the 
larva. The inner row or ring of cells we will call the chorda-mesenchyme 
ring, because it is destined to produce the chorda and mesenchyme. In 
it we must include the small flattened cells, C7, D™-°, but not their sis- 
ter cells, 07°, D'®, which, though in contact superficially with endoderm 
cells, really belong, as their fate shows, in the second or outer ring. 

The chorda mother cells, all of which are included in the chorda- 
mesenchyme ring, are derived, as has been already stated, in part from 
the anterior and in part from the posterior quadrants. Those derived 
from the anterior quadrants are at this stage eight in number. They 
form the anterior segment of the chorda-mesenchyme ring (Fig. 71, 
a™, q™19 gi-18 gi-34, and the corresponding cells on the left of the 
median plane). The posterior chorda cells are only two in number 


246 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


(c' 4, d™™, Fig. 71), one in the right and one in the left half of the 
embryo. They are now separated both from each other and from the 
anterior chorda cells. We shall see later how they are brought into 
contact with each other, in the median plane, and with the anterior 
chorda cells. 

The mesenchyme mother cells are also ten in number, but, unlike the 
chorda cells, they are derived chiefly from the posterior quadrants. They 
are A®!2\ its deep-lying sister cell, A*", d'*, D'*, and D"*, with the 
corresponding cells in the left half of the embryo, all indicated by a flat 
tint in the Figures.1 It will be observed that the mesenchyme fundament 
is made up of cells derived from both hemispheres and all four quadrants. 

The outer of the two rows of cells encircling the endoderm fundament 
will be called the newro-muscular ring. (Fig. 71. The cells are stippled.) 
It is interrupted at three points by mesenchyme cells of the inner ring ; 
in the middle line behind, by the small flattened cells, C7, D'®; on the 
right side, by A*”; and on the left side, by B32, Tt is thus divided 
into three portions, an anterior segment of eight cells, all descended 
from the anterior quadrants, and two latero-posterior segments, each 
composed of four cells, descended from one of the posterior quadrants. 
The anterior segment is composed purely of nerve mother-cells, which will 
form a considerable portion of the medullary plate. The other segments 
will form the entire longitudinal musculature of the larva, as well as a 
certain portion of the nervous system in the tail region. 

In the two rings of cells just described are included all save two of 
the descendants of the cells forming the equatorial band of the 48-cell 
and later stages. These two cells are D*” and C*”, situated at the 
posterior margin of the embryo (Fig. 71). . They form, in my opinion, 
definitive ectoderm. 

The remaining cells of the embryo number sixty-four, all descendants 
of the ectodermal group of the 48-cell stage. They will form definitive 
ectoderm, possibly also a portion of the medullary plate. 

One again notices in this stage the striking difference in rate of di- 
vision of the cells which he meets in passing from the vegetative toward 
the animal pole, a difference which made itself apparent as early as the 

1 Samassa (’94) identified the mesenchyme mother cells D78 and d‘!? (the cells 
8 and 9 of his Fig. 10) as nerve cells. In my preliminary paper I expressed a dif- 
ferent opinion, stating that they were mesoderm cells. Subsequent study has 
confirmed this view, but shown that I was wrong in stating, as I did, that they 
would contribute to the formation of “the longitudinal musculature of the tail.” 


That organ has, as I shall show, an entirely different and hitherto unsuspected 
origin. 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 247 


16-cell stage, and was foreshadowed still earlier by the internal constitu- 
tion of the unsegmented ovum. The endoderm fundament is in the sixth 
generation! (Plate XI. Fig..71, d*’, d®*, d®5, a®®, a®* and the corre- 
sponding cells in the left half of the Figure); the chorda-mesenchyme 
ring is chiefly in the seventh generation, though a single pair of its cells 
has recently passed into the eighth (D'*, D™®, d™-?, q™4, A812, 48-11, 
—the deep-lying sister cell of A*, not shown in the Figure, — a™4, a™-¥8, 
a), a, and the corresponding cells in quadrants B and () ; the cells 
of the neuro-muscular ring are all in the eighth generation, except a 
single pair which lingers in the seventh (D"-®, D*1*, D®”, D®8, A®-16, 
A’, AS-8, A*7, and the corresponding cells in quadrants B and C’); the 
ectoderm cells are all in the ninth generation, those nearest the animal 
pole having been the first to divide and pass into that generation. We 
notice in this stage, as in the earlier stages, a region of delayed division 
in the equatorial band at the posterior end of the embryo. 


B. HisToLoGicaL. 


Figures 63-70 (Plate X.) represent eight cross sections from a series 
through an embryo (not figured) a little more advanced in development 
than the one shown in Plate X. Figs. 61 and 62. The approximate 
position in the embryo of the sections figured is indicated by the hori- 
zontal lines on Figure 62. 

The differing stainability of cells at this stage, together with other 
histological peculiarities, serves already to distinguish the fundaments 
of the various organs with considerable precision. 

The endoderm cells (d*", Fig. 64; d®*, Figs. 65 and 66; a®8, d®&5, 
Fig. 67; a®®, Fig. 68; together with the corresponding cells in the left 
halves of these Figures) are, on account of their slow division, still very 
large. They are columnar in form, and contain large nuclei. Their 
cytoplasmic portion scarcely stains at all except in the region of the 
nuclei, being almost entirely taken up with close-packed yolk granules. 
A small amount of protoplasm staining a bright blue in hematoxylin 
extends out from either side of the nucleus in the long axis of the cell. 
In this small protoplasmic mass evidently lies an attraction sphere close 
up to the wall of the nucleus. The nuclei themselves contain numerous 
chromatic granules. 

The mesenchyme cells (D"%, Fig. 64; d™”, Figs. 65 and 66; A’, 
Fig. 67; together with the corresponding cells in the left halves of these 
Figures) are sharply distinguished from those of every other tissue by 


1 See the table on page 275. 


248 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


the very intense blue color which they take upon treatment with a 
hematoxylin stain. Their cytoplasm is not homogeneous at this stage, 
but contains numerous large dark-looking granules. The granules are 
undoubtedly yolk granules, and their dark appearance can often be seen 
upon close inspection to be due to an enveloping film of deeply staining 
protoplasm, which often extends out in radial processes, giving the whole 
a star-like appearance. This I believe to be caused by the progressive 
assimilation of the yolk granules and their conversion into protoplasm. 
In the case of the mitotic cell A’® (Fig. 67), and likewise of its mate in 
the left half of the same Figure, the characteristic mesenchyme stain- 
ing appears only in the more superficial portion of the cell, its deeper 
portion being loaded with yolk granules, which are still almost unat- 
tacked by the protoplasm. Consequently, when the approaching divis- 
ion is accomplished, the sister cells formed will differ from each other 
in appearance, the more superficial one being deeply stained, the other 
being stained scarcely at all. Subsequently, however, the yolk-laden 
cell will come to resemble in appearance its sister cell, and will have 
the same ultimate fate. The nuclei of the mesenchyme cells resemble 
closely in appearance those of the endoderm cells. In the case of ¢7-” 
and d‘” (Figs. 65 and 66), the nuclei are relatively small on account 
of recent division. 

The eight anterior chorda cells (a74, Fig. 68; a®*, a™°, and a’, 
Fig. 69 ; together with the corresponding cells in the left halves of these 
Figures) resemble closely in shape and stainability the endoderm cells. 
They are smaller, however, and contain nuclei, likewise smaller, with 
less conspicuous chromatic granules (omitted altogether in the Figures, 
as previously explained, to aid in readily distinguishing the chorda cells 
from those of other organs). 

The two posterior chorda cells (d7, Fig. 66; C7" [by mistake of 
engraver for ce"), Fig. 67) stain more deeply than the anterior chorda 
cells, resembling to some extent their sister cells d’” and ce? (Figs. 65 
and 66), from which they have recently been separated by division. 
However, they are many times smaller than their sister cells, and extend 
less deeply. This difference is connected with the oblique position of the 
spindles in the mother cells (see d°°, Fig. 60), a matter to which atten- 
tion was called in the discussion of the 64-cell stage. 

In the neuro-muscular ring the cells (stippled to distinguish them 
from those of other groups) have about the same histological character in 
both anterior (A®", Fig. 68 ; 4%“, Fig. 69; A®’, A’, Fig. 70; together 
with the corresponding cells in the left halves of these Figures) and pos- 


et - 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 249 


terior (D**, Fig. 65; D*”, Fig. 64; D’°, D*™, Fig. 63; together with the 
corresponding cells in the left halves of these Figures) portions. The cyto- 
‘plasm is finely granular and pretty homogeneous throughout, except in 
those portions of the cell most remote from the nucleus, where a certain 
amount of yolk is to be seen either unassimilated (Fig. 66, C8) or in 
process of assimilation (Figs. 68-70, A*'°, A*, A’, _A*", and their mates 
in quadrant B). In O7* (the mate of Din Fig. 63) both conditions 
are realized. Around the nucleus is the finely granular protoplasm, 
and in those portions of the cell most remote from the nucleus is the 
unattacked yolk. Forming a sharp line of boundary between the two 
is a zone in which assimilation is progressing, the yolk granules appear- 
ing here as large dark bodies. The color which the cells of the neuro- 
muscular ring assume is not so deep a blue as that of the mesenchyme 
cells; it is of a more grayish tint. 


B. Later STAGES OF GASTRULATION. 
(a) From the 112-cell to the 128-cell Stage. 


Figure 72 (Plate XI.) represents a dorsal view of a stage a little more 
advanced than the 112-cell stage shown in Figure 71. Sections (not 
figured) of this stage show (cf. sections of an older stage, Figs. 73-77) 
that the endoderm cells are in mitosis, the spindles being in all cases 
situated in an approximately horizontal position, so that after division 
the daughter cells will lie ina single layer forming a curved plate. The 
spindles are directed longitudinally in all the cells except two, viz. 
e°8 and d®® (cf. Plate X. Fig. 62), in which they lie transverse to the 
long axis of the embryo. 

Among the mesenchyme cells division has occurred in Dv’, C7 (ef. 
Figs. 71 and 74), the spindles standing vertically, as in the case of 
A’®, Bi (Plate X. Figs. 62 and 67), which divided earlier. Vertical 
spindles are also present in ec’, d™™ (cf. Plate XI. Figs. 71 and 75). 

The chorda cells are in the same generation as at the last stage, but 
the anterior ones are laterally compressed into a flattened or wedge shape, 
their thinner edges being directed backward. ‘They are situated at the 
anterior margin of the blastopore (Fig. 72). 

In the neuro-muscular band, two cells on each side of the blastopore 
(D*7, D*’, C*", C* 8) are seen to be in mitosis, their spindles being 
directed toward the centre of the blastopore. No evidence of division 
can be seen in any other cells of the embryo. It is therefore clear that 


the considerable advance in the process of gastrulation which is seen to 
VOL, XXVII.— NO. 7. 4 


250 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


have taken place since the stage shown in Plate XI. Fig. 71, has come 
about chiefly by an invagination, independent of cell division on the 
aboral surface, which has carried inward the endoderm and mesenchyme 
cells, has left at the margin of the wide-open blastopore the anterior 
chorda cells and the muscle cells, and has brought plainly into view, 
outside the neuro-muscular band, another row of cells from the ecto- 
dermal surface extending round the entire margin of the embryo. A 
certain number of cells at the anterior end of this new ring is destined 
to serve the same purpose as the anterior segment of the neuro-muscular 
band ; this fact is indicated in the figure by stippling. 

A clearer idea of the changes just sketched in outline may perhaps be 
had from an examination of cross sections. In Figures 73-77 are rep- 
resented five sections through the region of the blastopore of an embryo 
a little more advanced than the one shown in Figure 72. The approxi- 
mate position of the sections in the embryo is indicated by horizontal 
lines at the margin of Figure 72. In the endoderm cells the long 
deferred division leading to the seventh generation has at last been 
accomplished (Figs. 73-77). The endoderm cells accordingly number 
twenty, and their nuclei are greatly reduced in size on account of the 
recent division (cf. d®®, Fig. 66, Plate X., with d7™, d™-1°, Fig. 75, 
Plate XI.). The columnar form of the mother cells is retained by 
their descendants. 

It has been already stated that the spindles in the endoderm cells 
were at the recent division approximately horizontal in position. It is 
evident, therefore, that before the accomplishment of division the attrac- 
tion spheres must have shifted from the position which they were seen 
to occupy in Plate X. Figs. 66-68, for otherwise the spindles would 
have stood vertically, and a two-layered arrangement of the cells would 
have resulted, such as we shall see does occur in the case of the mesen- 
chyme cells. No mechanical explanation of this change in the position 
of the attraction spheres in the endoderm cells offers itself. The longest 
axis of the cells appears to be continuously the vertical axis, yet the 
spindles form in a direction transverse to this in every instance. Van 
Beneden et Julin’s (’86) Figures 1 ¢ and 2e also show spindles occupy- 
ing the short axis of the endoderm cells in the case of Clavelina. 

Considering now the mesenchyme cells we see (Fig. 74) that D *°,C™* 
(Fig. 71) have divided in such a manner that a small superficial cell is 
separated in each case from a many times larger deep-lying sister cell 
(cf. Fig. 74, D®-16, D*-15, 08-16, C8-15), A division similar in direction and 
in the inequality of its products is foreshadowed for the next anterior pair 
of mesenchyme cells (Fig. 75, e71, d™™), in which the spindles lie much 


a 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 251 


nearer to the superficial than to the deep ends of the cells. The most 
anterior mesenchyme cells appear in Figure 77 (A*2, A814, Be 12, Be12), 
They are descended from A‘, 57° (Plate X. Fig. 62), in which cells the 
division leading to the eighth generation occurred at a stage considerably 
earlier than this. (See Plate X. Fig. 67.) In this case also (A™*, B™) 
the spindles stood vertically and division was unequal, but the more 
superficial daughter cell was the larger, the deeper one being small and 
almost entirely filled with unassimilated yolk. (See Plate XI. Fig. 77, 
ABi2 8-11 R812, B8-11) 

The posterior chorda cells (d‘-", Fig. 77, and c7 in the left half of 
Fig. 76, not lettered) show no essential change since the last stage figured 
(cf. Plate X. Figs. 66 and 67). The anterior chorda cells do not appear 
in the sections figured ; they are still in the seventh generation. 

Division has been completed in D*:7, D*8, C*", O88 (Fig. 72), four of 
the neuro-muscular cells lying at the margin of the blastopore. (See 
Figs. 74-76, D®-®, D*4, D®-, D6 and the corresponding cells in 
quadrant C.) The other cells of the neuro-muscular ring and the entire 
ectodermal group have not been essentially modified since the last stage 
figured (Fig. 72). 

If the foregoing account is correct, the embryo, sections of which are 
shown in Figures 73-77, contains one hundred and twenty-eight cells, 
distributed as follows. 


Ventral hemisphere : — 


Ectodermal group : 
j . ectoderm. 
64 cells in the 9th generation 
(nerve cells. 

Equatorial band : 
2 ectoderm cells in the 9th generation. 


8 neuro-muscular cells in the 9th generation. 


10 a 2 cc 8th ee 
2 ee « ee 7th é 
8 mesenchyme ef 8th “ 
2 mesenchyme oe 7th <s 
32 — 


96 
Dorsal hemisphere : — 
2 mesenchyme cells in the 7th generation. 
10 chorda i: se sé 
20 endoderm a ‘ e 


252 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


(6) Closure of the Blastopore. 


In Figure 78 (Plate XI.) is shown a section parallel and slightly lat- 
eral to the median plane of an embryo a little more advanced than any 
thus far examined. It shows how the closure of the blastopore is 
coming about. 

The ectoderm cells on the ventral surface are seen to be smaller 
toward the anterior (left in the figure) than toward the posterior end 
of the embryo. They have evidently divided and passed into a later 
generation than those more posteriorly situated. This has caused them 
to spread over a greater surface, and has shoved the cells anterior to 
them farther around on to the dorsal surface of the embryo. In the 
anterior chorda cells, one of which is seen — in mitosis —in Figure 78 
(a’1", a®-18), division is nearly completed, the spindles standing about ver- 
tically. By the continued overgowth of the anterior lip of the blastopore, 
the more dorsally situated of the daughter cells in the chorda fundament, 
e. g. a®!", are carried posterior to their sister cells, e. g. a®-"* (cf. Fig. 79), 
and are finally entirely covered from sight by the nerve cells. They then 
form a plate of eight cells lying in the dorsal wall of the archenteron 
anterior to the blastopore. 

The endoderm cells of this embryo (Plate XI. Fig. 78) have under- 
gone no new divisions since the 128-cell stage (Figs. 73-77), though 
their nuclei have considerably increased in size, as is invariably the 
case during the resting period. More lateral sections than the one 
shown in Figure 78 exhibit spindles directed longitudinally in the 
mesenchyme cells A*?, 5°”? (cf. Figs. 71 and 77), and show that division 
has been completed in d’:¥, e7- (cf. Fig. 75). 

Sections through two other embryos, a little more advanced still in 
development, show in the muscle cells 07°, D™° (cf. Figs. 71 and 72) 
spindles directed forwards, inwards, and downwards, i. e. about toward 
the centre of the gastral cavity, a condition which is realized in the same 
cells at a corresponding stage in Clavelina. (See Van Beneden et 
Julin’s (86) Figs. la and 10.) These facts will aid us in interpreting 
sections of later stages. 

Figure 80 (Plate XI.) exhibits a dorsal view of a stage more advanced 
than any yet examined. The blastopore has greatly contracted (cf. 
Fig. 72) and now lies in the posterior half of the embryo. As gastru- 
lation has progressed, there has taken place an ingrowth of cells round 
the margin of the blastopore into the inner layer of the embryo. We 
have already seen how by this process the anterior chorda cells attain a 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 253 


position in the dorsal wall of the archenteron ; we may now observe that 
certain cells of the latero-posterior segments of the neuro-muscular ring 
are also involved in this ingrowth or invagination. At the stage shown 
in Figures 74-76 there was a double row of neuro-muscular cells on 
each side of the blastopore, now (see Fig. 80) the inner row is nearly 
covered from sight by the row of cells outside it. 

Already at the stage shown in Figure 72 the most posterior neuro- 
muscular cells (D"*, 07°) had moved from their originally lateral posi- 
tion toward the median plane (cf. Fig. 71). There they were destined 
presently to meet each other, covering over the small mesenchyme cells 
D>, C7 (cf. Figs. 71 and 72 with Fig. 80) ; now (Fig. 80) they or their 
descendants lie at the posterior angle of the blastopore, and are in turn 
being covered over by the more laterally and anteriorly situated neuro- 
muscular cells. 

The nerve cells anterior to the blastopore have increased considerably 
in number, perhaps through additions from the ectodermal group (ef. 
Figs. 72 and 80, also Figs. 78 and 79). 

Three sections from a horizontal series through an embryo of about 
the stage shown in Figure 80 are represented in Plate XI. Figs. 81-83. 
The series consists of thirteen sections 6.67 w thick, of which Figure 
81 represents the third, Figure 82 the fifth, and Figure 83 the seventh. 
The sections are a little oblique, and consequently strike the right and 
left halves of the embryo at slightly different levels. The left side of 
Figure 82 shows best the history of the mesenchyme cells since the last 
stage examined in detail (Figs. 73-77). Lateral to the small gastral 
cavity we find the sister cells B°-*, B%4, descendants of the common 
mother cell 6%: (Fig. 77). Evidence of the derivation of these two cells 
has been cited in the observation of a spindle longitudinally directed in 
the cell 6%? in two different embryos less advanced than this. 

Lateral to B*-*8 and 5*-*4 are the sister cells B%*!, B%?, descendants 
of Be (Fig. 71; cf. A®%", Fig. 77). They stain more faintly than 
B8 and 64, a distinction which, it will be remembered, existed 
between the respective mother cells 5%"! and B8? (Fig. 77). Though a 
spindle has in no embryo been directly observed in B*1, evidence of the 
sistership of 5°?! and 6%? (Fig. 82) exists in the still persistent inter- 
zonal filaments which stretch between their nuclei. This evidence is 
supported by the similarity of the cells in size and stainability. Poste- 
rior to the quartette of cells just discussed, the common descendants of 
B® (Plate X. Figs. 62, 67), are the two daughter cells derived from 
ce’ (Fig. 71), which was seen to be mitotic at an earlier stage (Fig. 75). 


254 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


It was predicted from the position of the spindle in this cell that the 
division would be unequal, the more centrally and dorsally situated 
daughter cell being the smaller. This smaller cell is represented by c**4 
(Fig. 82), but only the upper end of its large sister cell, c*?5, appears in 
this section. In Figure 83 (Plate XI.) we see the deeper portion of ¢*%%, 
which contains a nearly horizontal spindle. 

Posterior to the pair of cells just described are the descendants of C78 
(Fig. 71), the next to the hindmost of the mesenchyme cells in the left 
half of the embryo. In Figure 74 they were in the eighth generation 
(08-18, C8), One, the smaller, still remains in that generation (C**, 
Fig. 82), but its larger, more deeply situated sister cell has passed into 
the ninth generation, and is now represented by C*%*, C%, Figure 83. 
The direct evidence of mitosis has not been observed for the division 
here assumed, but very strong indirect evidence for it exists in the fact 
that at the last cell division in the mesenchyme cells, O*-* divided earlier 
than ec’ (cf. Figs. 74 and 75). Ifthe same order of division is followed 
in case of the daughter cells, division ought to occur earlier in C*- than 
in c’?8, But the latter cell is seen in Figure 83 to be in process of 
division ; therefore it is reasonable to suppose that at the same stage the 
former cell has already divided. 

The small posterior mesenchyme cells, C7, D5, lie one behind the 
the other deep down in the floor of the gastrula (Fig. 83), just posterior 
to the endoderm cells and overlaid by muscle cells, — for such the in- 
vaginated cells of the neuro-muscular ring become. 

These muscle cells have been crowded inward and downward at the 
posterior margin of the blastopore in consequence of the rapid contrac- 
tion of that opening. 

In the most posterior pair of muscle cells, viz. C7°, D’® (Fig. 71), 
mitosis was observed to occur, as already stated, at a stage earlier than 
this. The daughter cells arising from that division are readily recog- 
nized in O11, C*-12, and D®-™, D*4 (Fig. 83). The nucletof @* amd 
D*" lie in the section intermediate between those represented in Fig- 
ures 82 and 83. 

I am not able to declare with certainty the lineage of each of the 
other muscle cells in this series of sections, so I shall not attempt to 
point them out one by one. As a group, however, they are clearly 
distinguished from the ectoderm cells on the one hand, and from the 
mesenchyme cells on the other, by their large nuclei, their considerable 
size, and the peculiar stainability of their protoplasm. They resemble 
very closely in stainability the nerve cells lying anterior to the blasto- 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 255 


pore, but they are much larger than the nerve cells. They now lie lateral 
and posterior to the blastopore. (See Figs. 81-83 ; cf. Figs. 79 and 80.) 

The backward growth of the anterior lip of the blastopore has carried 
the crescent-shaped anterior chorda fundament (Fig. 71) from its origi- 
nal position to about the middle of the embryo’s dorsal surface (Fig. 81). 
It was seen in Figure 71 to consist of eight cells, which have now in- 
creased (Figure 78) to sixteen, and lie crowded together in two rows, one 
superposed above and overhanging the other (ef. Figs. 79 and 81. 

In Figures 62 (Plate X.) and 71 (Plate XI.) we saw that the two 
posterior chorda cells, viz. ec’, d‘"4, were separated from the ante- 
rior chorda cells by the mesenchyme cells, B':*, A™°, or their descend- 
ants. In Figure 82 we see that the descendants of B’*, A™® (viz. B%71, 
Be, 58, B%4, and the corresponding cells in quadrant A) during the 
process of invagination have been pushed down to the level of the other 
mesenchyme cells, allowing the anterior chorda cells to come into contact 
with the isolated posterior chorda cells (Fig. 81, c*4, c®-) above them. 
The posterior chorda cells were seen to be in the seventh generation in 
Figure 71 (ec, d’"). At the stage represented in Figure 81, there is 
good reason to believe that they have divided and passed into the eighth 
generation, since every other cell of the dorsal hemisphere is known to 
have done so previous to that stage; they are therefore represented by 
the cells c®-71, c&??, d°-71, and d°-”*, the last named cell being hidden from 
view in Figure 81 by the overlying muscle cell. 

The endoderm cells still remain in the eighth generation, and num- 
ber twenty. Their arrangement is made clear by an examination of 
Figures 81-83, in comparison with Figure 79, which represents a section 
near and parallel to the median plane of a slightly earlier stage. Four- 
teen of the twenty endoderm cells abut on the median plane, and six are 
placed laterally toward the anterior end of the embryo. The median 
double row of cells consists of 8" (Fig. 81), 8"? (Fig. 82), 57-7, 67-7, 
e716 T-14, ¢7-18 (Fig, 83), and the corresponding cells in the right half of 
the embryo. The nuclei do not appear in the centrally and posteriorly 
situated endoderm cells of Figure 83 because they lie in later (deeper) 
sections of the series, not figured (cf. Fig. 79). Only the narrow upper 
ends of the cells in question appear in Figure 83, which therefore gives 
no adequate idea of their size, but a correct idea of this may be had by 
an examination of Figure 79. The laterally situated endoderm cells 
are ce’ (Fig. 82), e7-!, e715 (Fig. 83), and the corresponding cells in 
the right half of the embryo. 

From a series of cross sections through an embryo in about the same 


256 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


stage as is represented in Figure 80, four sections have been selected to 
make more clear the relations of the fundaments of the various organs. 
(See Plate XII. Figs. 84-87.) Figure 84 represents a section just 
behind the blastopore (compare with it Plate XI. Fig. 73); in it the 
ectoderm is seen to have slightly overgrown from behind the most pos- 
terior muscle cells. (Compare Fig. 79, Plate XII.) Only one of the 
pair of small posterior mesenchyme cells (D’*, C7*) appears in the 
section; the other lies in the section just posterior to this. 

Mitosis is again setting in among the endoderm cells, as is shown by 
‘the spindle in d’-18; the next section anterior to this likewise shows 
spindles in the endoderm cells that are cut, c™* and d™"*. The spindles 
in each case (" and 74) are directed longitudinally, and in such a 
manner that the eight resulting cells will all lie in a single slightly con- 
cave layer. The consequence of these divisions will be a considerable 
elongation of the double row of endoderm cells at the posterior end of 
the embryo. 

It is worthy of note, though not shown in this series of sections, that 
at this division, as in the preceding and in subsequent ones, the spindles 
of the endoderm cells do not lie in the longest axis of the cells, which is 
the vertical. 

Figure 85 shows a section through the still open blastopore at its 
posterior margin. A comparison of this figure with Figures 72, 74, and 
75 (Plate XI.) shows that the ectoderm has grown rapidly in superficial 
extent through cell multiplication, and shoved the neuro-muscular cells 
C®:18, D8, inward to a position overlying their sister cells, O%4, D%™4, 
The small mesenchyme cell, C*-"* (cf. Plate XI. Fig. 74, D*®), is in pro- 
cess of division, following the lead of its large sister cell, C'*° (ef. Figs. 
74, D®5, and 84, D®-, D®-8). The mate of C*1®, viz, D*1® has already 
divided. One of its daughter cells is seen in this section (D**?, Fig. 85), 
the other lies in the next posterior section. The large mesenchyme cells, 
c8-8, q%28 (Fig. 85), are in mitosis (cf. Plate XI. Fig. 83). 

The section represented in Figure 86 encounters the blastopore farther 
forward than the one last described (Fig. 85), in its broader portion (ef. 
Plate XI. Figs. 72, 76, and 77). Here, too, the muscle cells have been 
crowded inward and partially invaginated ; C%> and D®™ overlie their 
sister cells, C%1® and D*1%, respectively. Of the posterior chorda cells 
only ce? and d* appear in this section. Their more laterally placed 
sister cells, c*?1 and d®-71, lie in the next posterior section (not figured), 
and at a slightly higher level (cf. Fig. 81, Plate XI.). 

Figure 87 (Plate XII.) represents the first section anterior to the 


ee ——  - . 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 257 


blastopore (cf. Plate XI. Fig. 80). On its depressed dorsal surface are 
seen six cells of the anterior chorda fundament, which is being rapidly 
covered over from the sides and anterior end by the ectoderm. Extend- 
ing deep down on either side of the chorda appears a deeply stained cell 
(stippled in the drawing) with large nucleus. These two are the most 
posterior cells of the medullary plate, which now lies at the dorsal surface 
of the embryo anterior to the blastopore, having been formed chiefly by 
the anterior segment of the neuro-muscular ring (cf. Plate XI. Fig. 80). 

The mesenchyme cells, A®**, 6*°4 (Fig. 87), are seen to lie on each 
side of the gastrula cavity (cf. Plate XI. Fig. 82) ; lateral to them lie the 
relatively small and faintly stained mesenchyme cells, A*”*, 6°77, The 
respective sister cells of those just mentioned, viz. A**8, 6% °8, 4%}, and 
5% (cf. Plate XI. Fig. 82), lie in the next two posterior sections (not 


figured). 


The stage next to be discussed differs in external appearance from that 
shown in Figure 80 chiefly, first, in the further contraction of its blasto- 
pore to a small aperture in the dorsal surface somewhat posterior to its 
centre ; secondly, in a slight elongation of the embryo and narrowing of 
its posterior end, foreshadowing the formation of the tail; and thirdly, 
in a slight depression of the medullary plate to form a neural or medul- 
lary groove (cf. Fig. 98). 

From a series of transverse sections through an embryo in this stage, 
five are represented in Plate XII. Figs. 88-92. Figure 88 (Plate XII.) 
shows a section posterior to the blastopore (cf. Fig. 98). It passes 
through the region of the small posterior mesenchyme cells, C7*, D**. 
Lateral or dorsal to them are seen four pairs of muscle cells containing 
large nuclei. The finely granular cytoplasm of these muscle cells takes 
a deep grayish blue stain in hematoxylin. Bounding the whole section 
is the uninterrupted ectoderm. 

The next anterior section, which has nearly twice the area of this, 
is likewise completely surrounded by ectoderm. 

The second section anterior to the one shown in Figure 88 is repre- 
sented in Figure 89 (Plate XII.). Two endoderm cells, the most pos- 
terior ones, appear in it. The small size of their nuclei indicates that 
they belong to a later generation than the endoderm cells seen in Fig- 
ure 84. Unquestionably they are in the eighth generation. To right 
and left of them appear two muscle cells, probably descendants of C7, 
Oe welate. XI: Fig. 73 (cf. Plate XI. Fig. 83,0? 4, €*™, DET and 
D**), Lateral to the muscle cells mentioned are seen in Figure 89 


258 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


(Plate XII.) mesenchyme cells, three on each side. Two of them are 
undoubtedly descendants of the mesenchyme cells C*, D®®, shown in 
Plate XI. Figs. 73 and 74, and represented in Plate XI. Fig. 83, and 
Plate XII. Fig. 84, by the cells C®-®, C?, D®-, and D®. Their nuclear 
condition shows that they have arisen from a recent division. Dorsal to 
the groups of cells already mentioned are seen in Figure 89 muscle cells 
extending up in a solid mass to the dorsal surface of the embryo. In 
the mid-dorsal surface of the section is a pair of cells, probably nerve 
cells, between which at an earlier stage lay the open blastopore. The 
periphery of the section is elsewhere bounded by ectoderm. 

Figure 90 (Plate XII.) represents the second section anterior to that 
shown in Figure 89. It passes through the widest portion of the blasto- 
pore. The only other section of the series which passes through the 
blastopore is the next preceding one, in which the blastoporic opening 
is extremely narrow, in fact, scarcely more than a slit. Figure 91 
shows the first section anterior to the blastopore. In it we see a plate 
of seven cells (cd.) belonging to the anterior chorda fundament and form- 
ing the roof of the archenteron (cf. Fig. 81). In Figure 90 we find the 
posterior chorda cells (ed.) lateral to the blastopore (cf. Plate XI. Fig. 81, 
c®2 and c®1), Ventral to the chorda cells in Figures 90 and 91 are 
the mesenchyme cells descended from c**, c*-*4, d*:8 and d%* (cf. 
Plate XI. Figs. 82 and 83). 

Dorsal to the chorda cells in Figure 91 are four cells unquestionably 
nervous, the two lateral ones being large and in mitosis, the other two 
small, evidently produced by recent divisions. In the next anterior 
section (not figured) the two lateral mitotic nerve cells again appear ; 
completely filling the space between them are four small nerve cells 
similar to the two seen in Figure 91. A medullary groove is thus clearly 
formed anterior to the blastopore, and the four cells dorsal to the chorda 
fundament in Figure 91 evidently are only lateral backward prolonga- 
tions of the medullary plate. The two large cells at the margins of the 
blastopore in Figure 90 are probably O* and D** (cf. Fig. 86) ; their 
deeper lying sister cells C®?® and D*”* have been carried into the more 
posterior sections by the crowding backward of the chorda cells and the 
elongation of the embryo. 

Figure 92 (Plate XII.) represents the third section anterior to the 
one shown in Figure 91. The medullary plate and chorda are here 
represented each by four cells. The mesenchyme cells visible on each 
side of the archenteron are A”, 6%:?8, A%71, and B*.7! (cf. Plate XI. 
Fig. 82). The cells A®*4, B*4, A®-, and B°-* (cf. Fig. 82) lie in the 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 259 


next two anterior sections. Of the eight cells mentioned, .A%%3, 49-34, 
Be, and 6*4 (cf. Fig. 82) are all in mitosis, but the four more laterally 
situated and smaller ones are still quiescent. 

Considering as a whole the mesenchyme of this embryo, we see that it 
consists of two lateral bands which have elongated with the elongation 
of the embryo. They now extend through ten different sections from 
near the anterior end of the embryo to a region posterior to the blasto- 
pore (Fig. 89). The muscle cells, on the other hand, are gathered into 
a pretty compact mass at the sides of and posterior to the blastopore 
(Figs. 88-90). 

In the subsequent stages of development the portion of the embryo 
lateral and posterior to the blastopore will be rapidly drawn out to 
form the tail of the larva, while the portion anterior to the blastopore 
will form the trunk. This will not come about, however, without a con- 
siderable shifting of cells from one portion into the other, for the chorda 
cells, which now lie anterior or lateral to the blastopore, must in large 
part pass into the tail, while the mesenchyme cells, which are more ven- 
trally located, and some of which now extend behind the blastopore, 
will all pass forward into the trunk region. 

An examination of Figure 98 (Plate XII.) may help to give a clearer 
idea of the stage just described. This figure shows a section made 
nearly parallel to the sagittal plane, but a little to one side of it, through 
an embryo slightly older than the one last under discussion (Figs. 88— 
92). The anterior chorda fundament, it is seen, has been carried back 
beyond the middle of the.embryo’s dorsal surface. The muscle cells 
have been forced backward and downward into a nearly vertical po- 
sition behind the blastopore, and are nearly covered over with ectoderm 
(cf. Fig. 93). 

Numerous cell divisions have recently occurred in the ectoderm, and 
the number of endoderm cells has also plainly increased. A very marked 
elongation of the embryo has attended these divisions. Several cells in 
the medullary plate are also dividing. On account of the slight obliquity 
of the plane of sectioning, the small posterior cells C%°, D™5 (ms’chy.), 
do not actually appear in this section as represented, but have been pro- 
Jected there from the adjacent section. In that section the endoderm 
extends back in a double row of cells into contact with C75, Dv5, as at 
the stages shown in Plate XI. Figs. 78 and 79. 

In Figures 93-97 (Plate XII.) are represented five cross sections 
through an embryo in about the same stage as is shown in Figure 98, 
The approximate position of the sections in the embryo is indicated on 


260 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


Figure 98 by the five vertical lines 93-97. Figure 93 shows a section 
posterior to the blastopore. It passes through one (ms’chy.) of the small 
posterior mesenchyme cells, D’*, 07° (cf. Fig. 88), the other lying in the 
next section posterior to this. The interior of the section is filled with a 
solid mass of muscle cells, or more properly nerve cells and muscle cells ; 
for it is highly probable that the four most dorsally situated of these 
cells, which form a group not quite covered in by the ectoderm, are to be- 
come part of the nerve cord of the tail (cf. Plate XIII. Figs. 99-101, x.). 
However, they are not distinguishable in histological characters from the 
more laterally and ventrally situated cells of the section. Cell division 
has recently occurred in the ectoderm, which plainly is soon to cover in 
completely the nerve cells in this region of the embryo. The muscle 
cells have evidently been reduced in size by division since the stage 
shown in Figures 88 and 89. 

The second section anterior to this is shown in Figure 94. It is the 
only section of the series which passes through the blastopore, now 
reduced almost to a slit. 

The blastopore is bordered on each side dorsally by a large nerve (?) 
cell, n. (cf. Fig. 90). Ventral to the nerve cells lie the posterior chorda 
cells, ed., lateral and still ventral to which are muscle cells. The most 
posterior pair of endoderm cells lies underneath the open blastopore, and 
a single small mesenchyme cell lies deep down in each half of the section. 

The second section anterior to the blastopore is shown in Figure 95; 
the second section anterior to that, in Figure 96; and one situated still 
two sections farther forward, in Figure 97. 

In Figure 96 the medullary plate is not at all depressed at its centre ; 
it consists of four large cells closely packed together and columnar in 
form. In Figure 97 the medullary plate is not even flattened, but con- 
forms to the evenly rounded contour of the embryo in that region. It 
consists of six cells sharply distinguished from the cells of the ectoderm 
in stainability, though the size of the more lateral ones is not materially 
different from that of the ectoderm cells. The chorda plate has dimin- 
ished to a breadth of only three cells in Figure 96, and is entirely want- 
ing in Figure 97, where endoderm cells occupy the space dorsal to the 
archenteron underneath the medullary plate. The mesenchyme bands 
cover considerable area in Figure 96, but are reduced to a single cell on 
each side of the body in Figure 97, from which it is seen that in this 
region the interior is nearly filled with a solid mass of endoderm. The 
section represented by Figure 97 lies well toward the anterior end of the 
embryo, as is indicated by the rapidly diminishing size of the sections. 


= iy cael, Teenie 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 261 


To summarize our observations on the series of sections Just examined 
(Figs. 93-97) : — 

(1) The fundament of the nervous system consists of a medullary plate 
extending from near the anterior end of the embryo to the blastopore, 
and continued backward by cells lying on each side of the blastopore 
and along ‘the line where the lips of the blastopore have fused. The 
transformation of the medullary plate into a medullary groove proceeds 
from the blastopore forward. 

(2) The chorda fundament consists of a plate of cells immediately 
underneath the medullary plate, but extending neither so far forward 
nor so far backward in the embryo. A part of it lies on each side of the 
blastopore, but the larger part is anterior to the blastopore. 

(3) The mesenchyme extends in two lateral bands from the region of 
the blastopore forward through about two thirds of the extent of the 
embryo anterior to the blastopore. 

(4) The muscle cells lie principally posterior to the blastopore in a 
pretty compact mass. They extend no farther forward than the first 
section anterior to the blastopore. 

(5) The endoderm consists of a double row of large cells ventrally 
situated extending from the first section behind the blastopore through 
the next five anterior sections ; it then broadens out and occupies nearly 
the whole inner layer of the embryo, both dorsally and ventrally, anterior 
to the chorda fundament. 


C. SumMMARY ON GASTRULATION. 


1. In the gastrulation of Ciona two processes can be distinguished : 
(a) a progressive invagination of the cells on the dorsal surface of the 
embryo, beginning at its centre; (6) a concomitant overgrowth of cells 
from the ventral side of the embryo, caused by more rapid cell division 
in that region. The overgrowth is greater at the anterior than at the 
posterior end of the embryo, because cell division proceeds more rapidly 
at the anterior end. 

2. Early in the process of gastrulation one can recognize a ring of 
cells encircling the blastopore peculiar in their stainability, forming the 
common fundament of the nervous system and the longitudinal muscu- 
lature of the larva.t_ Anterior to the blastopore the ring broadens out 


1 The existence of this peculiar ring of cells was first pointed out by Van Bene- 
den et Julin (’86) in the case of Clavelina; but these authors made the mistake of 
regarding it as exclusively nervous. 


262 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 


to form the medullary plate. Those cells of this newro-muscular ring 
which lie on each side of and posterior to the blastopore are for the 
most part invaginated, and form the entire longitudinal musculature of 
the tail. Some of them, however, form the most posterior portion of 
the nerve cord. 

3. Lying just within the margin of the blastopore, and €ncircled by 
the neuro-muscular ring, is another ring of cells, interrupted at the pos- 
terior end of the embryo only. Its anterior portion gives rise to the 
greater part of the chorda ; its remaining (lateral) portions produce the 
mesenchyme or trunk mesoderm, besides contributing to the chorda a 
single cell at each lateral margin of the blastopore. The descendants 
of these two chorda cells meet in the median plane at the closure of the 
blastopore. They form the most posterior portion of the chorda. 

We may regard the chorda-mesenchyme ring as being completed mor- 
phologically by the two small sub-chordal mesoderm cells 07°, D™, which 
have been wedged in between the most posterior cells of the neuro- 
muscular ring. Like the other cells of the chorda-mesenchyme ring, 
they lie in contact with the endoderm cells on one side, and with cells of 
the neuro-muscular ring on the other. Ultimately they probably form 
mesenchyme in the tail region. Possibly by a ccenogenetic reduction 
in size to their present minute dimensions, a gap has been left on each 
side of the embryo between them and the lateral portions of the chorda- 
mesenchyme ring. This change may have attended a ccenogenetic 
lengthening of the posterior end of the organism to subserve locomotion. 
There is evidence from other sources that the trunk of Ascidians formerly 
extended farther back into what is now the tail region of the larva. At 
that time the mesenchyme also probably extended farther back, and the 
chorda-mesenchyme fundament was in ontogeny, as we suppose it to 
have been in phylogeny, an uninterrupted ring. 

4. The blastopore, at first widely open, closes more rapidly from the 
anterior margin and from the sides than from behind. Consequently it 
comes to lie in the posterior portion of the dorsal surface of the embryo, 
and is triangular in form. The right and left sides of the triangular blas- 
topore, however, fuse from behind forward, beginning in the region of the 
pair of small, flattened mesoderm cells, C7*, D’®. Along the line of union 
of the lateral lips of the blastopore lies superficially on each side of the 
median plane a row of nerve cells. These are subsequently covered in 
by ectoderm from the sides and from behind, and form the posterior por- 
tion of the nerve cord. Underneath them, and at first not distinguishable 
from them in histological characters, are other cells, likewise derived from 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 263 


the posterior portion of the neuro-muscular ring ; these are destined to 
form the longitudinal musculature of the tail. The medullary plate, 
which produces the entire nervous system of the trunk region, lies wholly 
anterior to the region of “‘concrescence” of the lips of the blastopore. 

5. The posterior margin of the blastopore does not grow forward over 
the blastopore covering in the medullary canal as described by Van 
Beneden et Julin (’86) in the case of Clavelina.? 

6. I heartily concur in Samassa’s (94) conclusion that there is no 
rotation of axes during the gastrulation of Ciona, such as Korschelt u. 
Heider (93), on theoretical grounds, conjectured might occur in Ascid- 
ians. Their hypothesis is, so far as I know, entirely unsupported by 
observation. 


4. Formation of the Larva. 


The further changes which the embryo undergoes in its transformation 
into the larval tadpole will be understood from an examination of Fig- 
ures 99-105 (Plate XIII.), which represent seven sections through an 
embryo with completely closed blastopore. Figure 99 shows the third 
section (in passing from behind forward) of the series ; it contains about 
half a dozen muscle cells and four nerve cells, surrounded by an epithe- 
lial layer of ectoderm. ‘The first section of the series shows merely the 
ectoderm cut tangentially; the second contains six muscle cells sur- 
rounded by the ectoderm, but no nerve cells or chorda. The four nerve 
cells in Figure 99 show precisely the same arrangement as is found later 
_in across section of the tail of the larva. (See the four cells at the right 
of ed. in the right portion of Fig. 106.) 

In Figure 100 (Plate XIII.) the number of nerve cells (seven) is seen 
to be increased, and the chorda makes its appearance as a group of seven 
cells ventral to the nerve cells. 

In Figure 101 (Plate XIII.) the nervousand chorda fundaments 
appear about as in the section shown in Figure 100, but underneath the 
chorda is seen a group of four small mesoderm cells, the descendants of 
DD, CO" (Plate XII. Fig. 88), which have at last divided. Just ante- 
rior to them in the embryo (Figs. 102 and 103) extends the double row 
of caudal endoderm cells. As I have already suggested (page 262), the 
subchordal mesoderm cells (Fig. 101) probably have the same fate as 

1 The authors mentioned were doubtless led into this mistaken interpretation by 
identifying as nerve cells the muscle cells which lie behind the blastopore at the 


time of its closure. (See their Figs. 1a,1c, 2c, 8a, Pl. VII. These figures are 
reproduced in Korschelt u. Heider’s (93) Figs. 741 A, 741 B, 742 B, and 745 B, 


respectively.) 


264 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


the caudal endoderm cells, i. e. are resolved into mesenchyme at a later 
stage. 

Figure 102 (Plate XIII.) represents a section through what probably 
was the region of the blastopore. In it is seen the most posterior pair 
of endoderm cells (cf. Plate XII. Fig. 94). Since the stage last exam- 
ined (Plate XII. Figs. 93-97) the chorda cells have closed together into 
a single plate in this region, and the chorda fundament has grown farther 
back in the embryo. The nerve cells which lay at each side of the 
blastopore (Plate XII. Fig. 94) have also met in the median plane to 
form a single plate, which is now closing into a canal. A real canal is 
never formed posterior to the blastopore, though the nerve cells in that 
region potentially form one. 

Figure 105 (Plate XIII.) represents the second section anterior to the 
one shown in Figure 102; Figure 104, the second anterior to that ; and 
Figure 105, the fourth anterior to that. It will be seen that the muscle 
cells which in the series last examined (Figs. 93-97) were aggregated 
chiefly behind the blastopore, have now extended themselves not only 
posterior, but also anterior, to the blastopore. They extend as far for- 
ward as the next section in front of the one represented by Figure 103, 
i.e. through three sections anterior to the blastopore. They have 
pushed before them the mesenchyme, which in this series first appears 
in the section shown in Figure 103. The chorda fundament has mean- 
while moved toward the posterior end of the embryo. It now extends 
two sections behind the blastopore and overlies the small posterior me- 
senchyme cells (Fig. 101, cf. Plate XII. Fig. 93). “Accompanying the 
changes just mentioned, has come a diminution of the diameter of the em- 
bryo at its posterior end, which is already elongating to form the tail 
region. 

The mesenchyme extends forward of the section shown in Figure 103 
through six sections. The medullary plate extends forward two or three 
sections farther still. The endoderm consists of a donble row of cells 
extending forward underneath the chorda as far as the section seen in 
Figure 104, in which four endoderm cells are found; the arrangement 
there shown has been derived from that shown in Plate XII. Fig. 96, 
and still earlier in Plate XII. Fig. 91, by the meeting in the median 
plane underneath the chorda of the more laterally placed endoderm cells, 
Later, these four cells, or their descendants, will rnove apart so as to en- 
close between them the lumen of the posterior portion of the digestive 
tract. Anterior to the section shown in Figure 104 the endoderm 
rapidly increases in amount, while the chorda and mesenchyme diminish. 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 265 


In the region shown in Figure 105, it fills the entire interior of the 
section. 

In Figure 106 (Plate XIII.) is shown a section through an embryo 
in which the tail is already recognizable as a distinct portion of the 
embryo, though it has not yet reached anything like its maximum length. 

It is curved ventrad under the trunk, so that the section passes 
transversely through both trunk and tail. The section passes through 
the trunk in the brain region, but intersects only one of the mesenchyme 
bands, the other one not extending so far forward in the embryo. The 
endoderm cells are seen to have arranged themselves round a potential 
lumen in the form of an epithelium. However, they still lie two deep 
in places. Their shape is clearly becoming columnar. 

In the tail region appears the chorda, now transformed into a single 
row of flattened, disk-shaped cells, rapidly becoming vacuolated. They 
form an axial rod extending through the entire tail region and the pos- 
terior portion of the trunk. Dorsal (right in the Figure) to the chorda 
lies the nerve cord of the tail, composed in cross section of about four 
small cells. 

Ventral to the chorda is the sub-chordal endoderm strand consisting 
of a double row of cells (en’drm.). On each side of the chorda are seen 
in the section about three muscle cells. 


SUMMARY ON FORMATION OF THE LARVA. 


1. The nerve cord in the limited region of concrescence of the lips of 
the blastopore is covered over by the ectoderm first at its posterior end 
and then successively in its more anterior regions, following the course 
of conerescence. The nerve cells in that portion of the embryo never 
form a real canal, but only a potential one. They are arranged in a 
solid strand, which usually shows in cross section four cells placed round 
a common centre, the potential canal. 

The medullary plate arises wholly anterior to the blastopore. At the 
time when the blastopore is about to close, the medullary plate has come 
to extend over a great part of the length of the embryo, and has sunk 
down in the form of a shallow grove deepest at its posterior end, the 
anterior margin of the blastopore. When the blastopore closes, it be- 
gins to form a canal. This process, like the fusion of the lateral mar- 
gins of the blastopore, advances from behind forward. 

2. Beginning shortly before the closure of the blastopore, a rapid 
elongation of the embryo takes place, accompanied by a considerable 
change in its form and a rearrangement of the cells composing some of 


VOL. XXVII. —NO. 7. 5 e 


266 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


its organs. The posterior end of the embryo, which toward the comple- 
tion of gastrulation was broader than the anterior end, becomes narrower 
and narrower, and ultimately forms the tail, which is curved ventrad 
around the trunk of the embryo within the egg membranes. 

Before the closure of the blastopore the chorda is a plate of cells lying 
in the dorsal wall of the archenteron, anterior and lateral to the blasto- 
pore. The portions lateral to the blastopore meet in the median plane 
when the blastopore closes. The chorda fundament then elongates, 
owing to a shoving together of its cells from each side, ‘like a pack of 
cards in shuffling” (Van Beneden et Julin), until they form, instead of 
a plate, a single median row of disk-shaped cells arranged one behind 
another like a row of coins and reaching backward underneath the nerve 
cord to the extreme posterior end of the embryo. Anteriorly it termi- 
nates not far from the middle of the trunk region. 

The muscle cells, which originally lay on each side of and behind the 
blastopore, extend themselves a single cell deep in two bands, one on 
each side of the chorda throughout its entire length. 

The mesenchyme cells originally formed the lateral portions of the 
chorda-mesenchyme ring. As the blastopore gradually closed, they were 
thrust down to a deeper level than the muscle cells, and forward. UIti- 
mately they come to lie wholly in the trunk region, chiefly in its pos- 
terior portion, in two pretty compact lateral masses of small deeply 
stained cells, two or more layers deep. At a still later period, these 
lateral masses are resolved into migratory cells, i. e. blood corpuscles, 
mantle cells, ete. 

Before the closure of the blastopore the endoderm forms the entire 
lining of the archenteron in its most anterior portion, where its lumen is 
almost obliterated. Farther back the chorda forms the dorsal wall of 
the archenteron, the mesenchyme cells forming its sides, the floor only 
being occupied by the endoderm cells. In the region where the blastopore 
closes, the endoderm cells occur only as a double row ventrally situated 
along the median line. 

This double row is extended back in the larva underneath the chorda 
throughout almost the entire length of the tail, forming a “subchordal 
endoderm strand,” which is ultimately resolved into wandering cells, 
or perhaps utilized as food material by the mesenchyme cells of the 
trunk region. At the posterior end of this caudal endoderm strand lie 
the small mesoderm cells which Van Beneden et Julin mistakenly 
included in the nervous fundament. These cells are to be regarded as 
the most posterior constituents of the original chorda-mesenchyme ring. 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 267 


Like the cells of the endoderm strand just anterior to them, they prob- 
ably become wandering cells. 

In the posterior portion of the trunk region, where before the closure 
of the blastopore the endoderm strand broadens out into a plate of four 
or more cells, the more laterally placed endoderm cells move dorsad 
at the closure of the blastopore, and meet in the median plane under- 
neath the chorda. In this way the endoderm of the trunk region is 
converted into a closed vesicle, pear-shaped and broadest in its anterior 
portions ; at its posterior end it is overlaid by the chorda and flanked on 
each side by the mesenchyme. 


VII. DISCUSSION OF SOME THEORETICAL 
QUESTIONS. 


The facts presented in the foregoing pages have a certain bearing 
on several questions of general interest. Of these I shall make brief 
reference to, — 1. The origin of the germ layers of Chordates; 2. The 
Ceelom theory ; and 3. The ancestry of Chordates. 


A. Origin of the Germ Layers of Chordates. 


According to the generally accepted doctrine of Haeckel, all the higher 
metazoa are ultimately derived from a simple cup-shaped or sac-like 
ancestor composed of two cell layers, an inner and an outer, continuous 
with each other at the margin of the cup or sac. The two cell layers are 
called the primary germ layers. The outer layer is known as the pri- 
mary ectoderm ; the inner, as the primary endoderm. Among the Chor- 
dates, this supposed ancestral condition is most nearly realized in ontogeny 
in the case of Amphioxus. The homologues of its inner and outer germ 
layers are traced by embryologists through all the groups of the chor- 
date phylum. A third or middle layer, derived from one or both of the 
others, makes its appearance between the two primary germ layers in all 
the higher Metazoa. Whether this middle layer, or mesoderm, is homol- 
ogous throughout the different groups of Metazoa is one of the most 
difficult and disputed questions in the whole realm of comparative em- 
bryology. Into this question I do not propose to go in this paper; I 
shall confine my attention to the mesoderm of Chordates. 

It is commonly believed that the mesoderm of Chordates is derived 
entirely from the inner germ layer, which is accordingly often referred to 
as mes-endoderm. With this view, however, my observations on Ciona 


268 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


force me to take issue. In an earlier part of this paper it has been 
shown that during the process of gastrulation in Ciona there is a progres- 
sive ingrowth of cells around the blastopore from a position in the outer 
to a position in the inner layer of the gastrula. Whether, therefore, 
we shall include a particular cell in the primary ectodorm or primary 
endoderm depends on whether we consider the embryo at an earlier or 
a later period in the process of gastrulation. Lwoff (94) has recently 
stated, and it seems to me on excellent evidence, that in Amphioxus 
and all Vertebrates there occurs in the formation of the germ layers an 
ingrowth of cells from the outer to the inner layer of the embryo, very 
similar to that which I have observed in Ciona. He accordingly distin- 
euishes what we may call a primary invagination of the cells destined 
to form chiefly the alimentary tract from a secondary invagination 
involving the cells destined to form the chorda and a portion of the 
mesoderm, viz. the musculature. The matter seems to me of sufficient 
importance to warrant the quotation of the author’s own words. Speak- 
ing of Amphioxus he says :— 

“Tech bin also zam Schluss gekommien, dass die dorsale Wand der Gas- 
trulahihle, die ich als dorsale Platte bezeichnen will, von den Ektoderm- 
zellen gebildet ist. Dieses Ergebniss ist von sehr grosser Bedeutung. Wie 
die weiteren Entwickelungsstadien lehren, stellt diese dorsale Wand 
die Anlage der Chorda und des Mesoderms dar, indem aus der mit- 
tleren Zellenpartie derselben die Chorda, aus zwei seitlichen Theilen 
das der Chorda anliegende Mesoderm entsteht, aus welchem, wie be- 
kannt, die Muskelelemente entstehen. Dies zeigt, das die Chorda und 
das anliegende Mesoderm aus einer ektoblastogenen Anlage entstehen, die 
urspriinglich als eine zusammenhingende Platte (dorsale Platte) er- 
scheint. Was die eigentlichen Entodermzellen betrifft, welche die tibrige 
Wandung der Héhle bilden, so will ich hier in Ktirze vorbemerken, dass 
sie jederseits einige an die dorsale Platte angrenzende Zellen als ihren 
Beitrag zur Bildung des Mesoderms abgeben; die Rinder des tibrig- 
gebliebenen Entoderms wachsen unter den seitlichen Mesodermanlagen 
nach der Mittellinie zu, vereinigen sich unter der Chorda und bilden 
auf solche Weise den Darm. 

“ Das Hauptergebniss dieser Untersuchungen ist, dass die Hinstiilpung 
bei Amphioxus keineswegs als eine einfache Gastrulation zu betrachten ist, 
wie es bisher angenommen. Ls sind vielmehr hier zwei verschiedene Pro- 
cesse zu unterscheiden: erstens die Hinstiilpung der Entodermzellen, aus 
denen der Darm entsteht ; zweitens die Hinstiilpung der Ektodermzellen vom 
dorsalen Umschlagsrande aus, welche die ektoblastogene Anlage der Chorda 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 269 


und des Mesoderms bildet. Die Einstiilpung der Entodermzellen ist als 
Gastrulation zu betrachten. Es ist ein palingenetischer Process, den die 
Chordaten von ihren Vorfahren ererbt zu haben scheinen, wo dieser Pro- 
cess gleichmissig und radial symmetrisch vor sich ging, wie es sich bei 
einigen wirbellosen Thieren beobachten lisst. Die Kinstiilpung der Ekto- 
dermzellen ist dagegen als ein coenogenetischer Process zu betrachten, der 
mit der Bildung des Darmes nichts zu thun hat und durch den die Bil- 
dung der ektoblastogenen Anlage der Chorda und des Mesoderms eingeleitet 
wird. Wie in den folgenden Abschnitten gezeigt werden soll, lassen sich 
diese zwei Processe—die Bildung des Darmes und die Bildung der 
ektoblastogenen Anlage der Chorda und des Mesoderms — auch in der 
Entwickelung aller Wirbelthiere von einander unterscheiden.” (Sepa- 
rate, pp. 11, 12.) 

Whether Lwoff is right in including the chorda and the greater por- 
tion of the mesoderm of Amphioxus and the Vertebrates in the second- 
arily invaginated part of the inner germ layer, I do not attempt to say. 
That question must be decided by an examination of the forms on which 
he made his observations. However, in Ciona, at least, the cells which 
are destined to form chorda and mesenchyme (chorda-mesenchyme ring 
must be regarded as taking part in a primary invagination along with 
the definitive endoderm. But plainly a very important part of the me- 
soderm, viz. the cells which form the longitudinal musculature, is carried 
into the inner layer by a secondary invagination. The secondarily 
invaginated cells are derived from the posterior segments of the neuro- 
muscular ring. At the beginning of gastrulation they clearly lie in the 
outer layer of the gastrula, but at the conclusion of gastrulation they lie 
within the margin of the blastopore. 

Accordingly, I regard the definitive endoderm fundament and the en- 
circling chorda-mesenchyme ring as constituting the primary endoderm 
in Ciona. In the primary ectoderm, I would include the neuro-muscular 
ring and the “ectodermal group’ 
the outer layer of the gastrula when the closure of the blastopore begins. 
If this view is correct, the mesoderm or middle germ layer of Ascidians 
must be regarded as derived in part from the primary endoderm and in 
part from the primary ectoderm. Lwoff reached a similar conclusion 
concerning the origin of the mesoderm in Amphioxus and the Verte- 
brates. In Ciona that part of the mesoderm which is derived from the 
outer germ layer produces the longitudinal musculature of the larva. It 


> 


of cells, both of which lie entirely in 


forms the whole of this tissue, and nothing else. Similarly in Amphioxus 
and the Vertebrates, Lwoff concluded that the ectodermal mesoderm 


270 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


formed the longitudinal musculature. It would seem, therefore, that 
the muscle plate of the mesodermal somite of Amphioxus is homologous 
with the muscle cells of the Ciona tadpole. Both in Amphioxus and in 
Ciona the muscle fundament arises from cells lying lateral to the chorda 
and derived from the primary ectoderm. In Amphioxus the muscula- 
ture, like the chorda with which it is intimately associated, becomes 
(coenogenetically ?) extended far forward to the anterior end of the 
trunk region; whereas in Ciona neither musculature nor chorda extends 
farther forward than about the middle of the trunk region. 

The mesoderm lateral to the muscle plates of Amphioxus seems to be 
the homologue of the mesenchyme of Ciona. Both are derived from 
the endodermal portion of the mesoderm. (Cf. the quotation from Lwoff, 
pages 268, 269.) 

My conclusions differ from those of Lwoff chiefly regarding the origin 
of the chorda. He considers this organ to be derived from the primary 
ectoderm in Amphioxus and the Vertebrates, whereas I regard it as 
formed in Ciona exclusively by the primary endoderm. I think that 
Lwoff has been led to include the chorda cells in the primary ectoderm 
chiefly because they are in Amphioxus (as in Ciona) smaller and clearer 
than the less rapidly cleaving endoderm cells. These criteria I regard 
as insufficient. Only a study of the cell lineage can give in any case 
a positive answer to the question whether the chorda cells at the begin- 
ning of gastrulation lie in the outer or the inner layer of the embryo. 

That a distinction is rightly made in the case of Ascidians between 
the two kinds of mesoderm which I have recognized, viz. musculature 
and mesenchyme, is unanimously agreed to by embryologists ; but the 
fact has been heretofore overlooked that these two kinds of mesoderm are 
derived from different fundaments early distinguishable both histologically 
and topographically, and that these fundaments should be regarded as 
derived from different primary germ layers. 

A minor point of theoretical importance is whether or not the chorda 
shall be regarded as a mesodermal organ. Lwoff does not so consider it, 
though he recognizes two facts which, it seems to me, would naturally lead 
one to that conclusion: the first, that in Amphioxus and the lower groups 
of Vertebrates the chorda is derived from a common fundament with 
what is universally regarded as mesoderm ; the second, that the chorda, 
like the undoubted mesoderm, comes to occupy a position between the 
inner and outer layers of the embryo. For these two reasons, which 
I have shown to exist also in the case of Ascidians, we must, to be con- 
sistent, regard the chorda as a mesodermal organ. 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS, 271 


Seeliger (’85), Davidoff (89), and Samassa (94) all state that the 
first equatorial plane of cleavage in the ascidian egg separates the two 
primary germ layers. According to my definition of the primary germ 
layers in Ciona, this is not true, for several of the cells composing the 
chorda-mesenchyme ring (included by me in the primary endoderm) are 
derived from the four ventral cells, which according to their view are 
exclusively ectodermal. The statement that the first equatorial plane of 
cleavage separates the two primary germ layers is equally untenable, if 
tested by the definition of primary germ layers accepted by the authors 
mentioned ; for they include in the primary endoderm the entire meso- 
derm, which I have shown to be derived ehiefly from the four ventral 
cells, which produce the definitive ectoderm. 


B. The Colom Theory. 


The brothers Hertwig (’81) divided the higher Metazoa into two 
groups according as the body cavity arises by a pair of outpocketings 
of the primary endoderm enclosing an enterocel between visceral and 
parietal mesoderm layers, or by a simple splitting or moving apart of 
cells in asolid mass of mesoderm, which is then said to enclose a 
schizocel. The Chordates were unhesitatingly placed by them among 
the Enteroccelians, and Amphioxus was cited as a typical example. The 
Tunicates were thus classed as Enteroccelians, though no one had ever 
observed in their ontogeny the formation of an enterocel. Van Bene- 
den et Julin (86) supplied the lack by their studies on Clavelina; but 
considerable doubt has been thrown on the accuracy of their observa- 
tions by the independent researches of Seeliger (85) upon an undeter- 
mined species of the same genus, and by those of Davidoff (’91) upon 
the identical species studied by Van Beneden et Julin. Neither Seeliger 
nor Davidoff could detect a trace of enteroceel formation in the ontogeny 
of Clavelina, and Davidoff was equally unsuccessful in finding an enteroccel 
in Distaplia. My own observations on Ciona are entirely in agreement 
with those of Seeliger and Davidoff on this point. Van Beneden et 
Julin, notwithstanding their belief that an enteroccel is formed in Ascid- 
ians, as well as in Amphioxus, rejected the classification of the brothers 
Hertwig on other grounds. 

Lwoff (94) has recently shown that in Amphioxus the cavities enclosed 
by outpocketings of the wall of the gastral cavity are evanescent struc- 
tures, and have nothing to do with the subsequently formed body cavity, 
which, as in all Vertebrates, arises by a wandering apart of mesoderm 


Dita BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


cells. He therefore concludes “ dass ein wahrer Enterocoelier unter allen 
Chordaten nicht existirt.” 

I am not able to criticise Lwoff’s conclusion from the vantage ground 
of personal investigation of Amphioxus, but his account bears internal 
evidence of careful and exact observation. He calls attention to a fact, 
shown by his figures, that, when the mesodermal pouches arise, spindles, 
if any are present in the mesoderm cells, invariably stand vertically to 
the evaginated layer of cells, foreshadowing an arrangement of the 
daughter cells in two layers. This is exactly the position which the spin- 
dles take during gastrulation in the mesenchyme cells of Ciona, but in 
no other cells of the embryo. The form of division in the mesoderm 
cells of Amphioxus at the period mentioned tends to obliterate the lumen 
of the mesodermal pouches, a result which, as Lwofi’s figures show, actu- 
ally comes about. A body cavity is formed only secondarily by the 
moving apart of the mesoderm cells which are arranged in solid masses, 
the protovertebre. 

Davidoff (91) likewise observed in the case of the compound Ascidian, 
Distaplia, that spindles stand vertically in the cells which give rise to the 
mesoderm at the time of the separation of the middle germ layer. He 
believes that the Tunicates can in no sense be regarded as Enteroccelians, 
and, further, that the distinction made by the brothers Hertwig between 
those Metazoa which possess a “mesoderm” and those which possess 
“mesenchyme ” is an artificial and unsound one. With these conclu- 
sions I entirely agree. 

Regarding Rabl’s (’89) distinction between “ gastral” and “ peristo- 
mal” mesoderm, my observations lead me to the same conclusion as has 
been expressed by Davidoff, “dass das peristomale Mesoderm der Ascidien 
sich im weiteren Verlauf der Entwicklung zum gastralen herausbildet, 
oder dass das gastrale Mesoderm urspriinglich peristomales Mesoderm ist.” 
O. Hertwig (92) draws a similar conclusion regarding Rabl’s distinction 
as applied to the Vertebrates. 

I should also state that both Lwoff and Wilson (94) find that the 
pole mesoderm cells described by Hatschek in the case of Amphioxus 
do not exist. Certainly nothing of the kind is found among Ascidians. 
Hence we may conclude that such cells are entirely wanting among 
Chordates. 


C. Ancestry of the Chordates. 


To determine the phylogenetic relationship of the Chordates to the 
other groups of Metazoa is a very difficult problem. Various solutions 


Se ee 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. Qo 


of it have been offered, but none is very generally accepted among 
zoOlogists. ‘The group is sharply marked off from all others by the 
possession of certain peculiar characters, such as the chorda, gill slits, 
and hypophysis. Among the higher Metazoa the Chordates seem to 
have no near relatives. 

An ingenious suggestion, which has gained considerable currency, is 
that a chordate is homologous with an annelid whose dorsal and ventral 
surfaces are reversed. This “annelid hypothesis” has been ably advo- 
eated by Dohrn (’75 and ’82-91) and Kisig (’87). An extensive adverse 
criticism of the hypothesis has been made by Brooks (’93). Is any light 
thrown on the question by the ontogenetic history of Tunicates? The 
evidence from that source seems to me chiefly negative. Recent studies 
of the embryology of Annelids and Mollusks show a truly marvellous cor- 
respondence between the developmental processes in these two groups ; 
it is even possible to refer back particular organs in both to homologous 
blastomeres, and to trace their differentiation through unmistakably sim- 
ilar processes. No doubt is left in the mind as to the close phylogenetic 
relationship of Annelids and Mollusks. The embryology of Chordates, 
however, follows an altogether different course, and is as unlike that of 
Annelids as the adult forms are different. 

It is possible that we must go as far down in the animal scale as the 
Celenterates to find an ancestor common to the Chordates and any other 
group of the higher Metazoa. The embryology of Tunicates seems to 
me to support this view. 

Brooks (793) has shown good reason for believing that all the principal 
groups of Metazoa arose as small, permanently pelagic forms, such as we 
find represented to-day, in a somewhat modified form, by Appendicularia 
in the case of Chordates. 

Amphioxus, because of its adaptation to life on the bottom, has prob- 
ably undergone considerable modification from the ancestral type. For 
example, the chorda has been extended forward to the extreme anterior 
end of the body to admit of the animal’s burrowing in the sand; a 
marked asymmetry of the body has also arisen, and its size has doubt- 
less greatly increased, calling for a metameric arrangement of its organs. 

The ascidian tadpole, too, has probably been somewhat modified by a 
great shortening of the free-swimming (ancestral) period of its exist- 
ence ; but here the changes have probably been restricted to a suppres- 
ston of certain processes or organs, so that those which remain are more 
certainly ancestral than those which occur even in Amphioxus. The 
post-larval history of Ascidians clearly exhibits a process of degeneration, 
which of course is wholly coenogenetic. 


274 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


VIII, CONCLUSIONS. 


1. In the maturation of the ascidian egg the polar globules arise at 
the vegetative pole, i.e. in the future endodermal portion of the egg. 

2. The archoplasms (attraction spheres) of the first cleavage spindle, 
and consequently of all subsequent spindles of the fertilized ascidian 
egg, are derived exclusively from the spermatazoon. 

3. The archoplasm (attraction sphere) is not an organ of heredity, 
since in sexual reproduction it is frequently derived from one parent 
only. 

4. Cleavage in the ascidian egg is bilateral from the very beginning. 
The course of cleavage is less variable in the egg of Ciona than in that 
of Amphioxus or the Vertebrates, and is predetermined by the internal 
constitution of the unsegmented egg. 

5. The first equatorial plane of cleavage does not separate completely 
the two primary germ layers, though it does separate definitive endoderm 
from definitive ectoderm. 

6. The fundaments of the principal organs are arranged in zones 
around the chief axis of the egg. 

7. The nervous system and the longitudinal musculature of the larva 
are derived from a common fundament, which is a (neuro-muscular) ring 
of cells encircling the margin of the blastopore. This ring of cells must 
be regarded as a part of the primary ectoderm. 

8. The chorda and mesenchyme (or trunk mesoderm) are derived from 
another ring of cells lying just within the margin of the blastopore. 
This ring of cells is to be regarded as a part of the primary endoderm. 

9. The mesoderm of Ascidians is therefore derived in part from the 
primary ectoderm, and in part from the primary endoderm. It is 
formed exclusively by cells of the two rings already mentioned, one of 
which belongs to each of the two primary germ layers. Recent careful 
observations indicate that likewise in Amphioxus and the Vertebrates 
the mesoderm is derived from both primary germ layers. 

10. The longitudinal musculature of the Ascidian tadpole is homolo- 
gous with that of Amphioxus; the mesenchyme of the Ascidian, with 
the mesoderm lateral to the muscle plates in Amphioxus. 

11. The chorda should be regarded as a mesodermal organ. 


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*TL99-$9 “TL99-8h | *T199-GE *TL99-FG *TL99-9T “1199-8 “T09-F “T199-G 


‘VNOIO JO HOVHNIT ITTHO AO HIAVL “XI 


ye a a. > n * ees * 2 ke + ey SSS 


276 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


LITERATURE CITED. 


Agassiz, L. 
49. Characteristics of new Specics from the Shores of Massachusetts. Proce. 
Amer. Assoc. Adv. Sci., Vol. II. pp. 157-159. 


Beneden, E. van, et Julin, C. 
’84. la segmentation chez les Ascidiens et ses rapports avec l’organisation de 


la larve. Arch. de Biol., Tom. V. pp. 111-126, Pl. Vil., VIII. 


Beneden, E. van, et Julin, C. 
’86. Recherches sur la morphologie des Tuniciers. Arch. de Biol., Tom. VI. 
pp- 237-476, Pl. VIL-XVI. 
Boveri, T. 
’88. Zellenstudien, Heft II. Die Befruchtung u. Theilung des Eies von As- 
caris megalocephala. Jena. Zeitschr., Bd. XXII. pp. 685-882, Taf. XIX—- 
XXIII. 


Boveri, T. 
90. Zellenstudien, Heft III. Ueber das Verhalten der chromatischen Kern- 
substanz bei der Bildung der Richtungskérper und bei der Befruchtung. 
Jena. Zeitschr., Bd. XXIV. pp. 314-401, Taf. XI-XIII. 


Brooks, W. K. 
93. The Genus Salpa,a Monograph with fifty-seven Plates. (303 pp., 47 Pls.) 
With a Supplementary Paper — The Eyes and Subneural Gland of Salpa— 
by M. M. Metcalf, pp. 305-371, Pls. XLVIII-LVII. Johns Hopkins 


Press, Baltimore. 


Castle, W. E. 
94. On the Cell Lineage of the Ascidian Egg. A Preliminary Notice. Proc. 
Amer. Acad. Arts and Sci., Vol. XXX. pp. 200-217, 2 Pl. 


Chabry, L. 
’87. Contribution & l’embryologie normale et tératologique des Ascidies sim- 
ples. Journ. Anat. et Physiol., Tom. XXIII. pp. 167-319, Pl. XVILI- 

XXII. 


Conklin, E. G. 
94. The Fertilization of the Ovum. Biol. Lectures, Mar. Biol. Lab., Wood’s 
Holl, [Vol. II.] pp. 15-35, 10 Figs. 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 211 


Davidoff, M. v. 
89-91. Untersuchungen zur Entwickluugsgeschichte der Distaplia magni- 
larva, Della Valle, einer zusammengesetzten Ascidie. Mitth. Zool. Stat. 


Neapel, Bd. IX. pp. 115-178, 533-651, Taf. V., VI., XVIII.-XXIV. 


Dohrn, A. 
'75. Der Ursprung der Wirbelthiere und das Princip des Functionswechsels. 
15 pp. Leipzig, 1875. 


Dohrn, A. 
8291. Studien zur Urgeschichte des Wirbelthierkérpers. Mitth. Zool. 
Stat. Neapel, Bd. I1I.-X. 


Eisig, H. 
’87. Die Capitelliden des Golfes von Neapel. Fauna u. Flora des Golfes von 
Neapel, Monogr. XVI. 


Fick, R. 
’'93. Ueber die Reifung u. Befruchtung des Axolotyleies. Zeitschr. f. wiss. 
Zool., Bd. LVI. pp. 529-614, Taf. XX VII.-XXX. 
Fol, H. 
'77. Sur la formation des ceufs chez les Ascidiens. Journ. d. Micrographie, 
Tom. I. p. 281. 
Fol, H. 


'84. Sur lceuf et ses enveloppes chez les Tuniciers. Recueil Zool. Suisse, 
Tom. I. pp. 91-160, Pl. VII., VIII. 


Guignard, L. 
91. Nouvelles études sur la fécundation. Ann. d. Sci. Nat., sér. VII., Bot., 
Tom. XIV. pp. 163-296, Pls. 9-18. 


Hertwig, O. 
’83. Die Entwicklung des mittleren Keimblattes der Wirbelthiere. 128 pp., 
9 Taf. Jena. 


Hertwig, O. 
‘92. Urmund und Spina bifida. Arch. f. mikr. Anat., Bd. XX XIX. pp. 353- 
503, Taf. XVI.-XX. 


Hertwig, O. und R. 
’81. Die Coelometheorie. 146 pp., 3 Taf. Jena. 


Kingsley, J. S. 
’83. Some Points in the Development of Molgula Manhattensis. Proc. Bost. 
Soc. Nat. Hist., Vol. XXI. pp. 441-451, 1 Pl. 


Kofoid, C. A. 
94. On some Laws of Cleavage in Limax. Proc. Amer. Acad. Arts and Sci., 


Vol. XXIX. pp. 180-200, 2 Pls. 


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Kupffer, C. 
70. Die Stammverwandtschaft gwischen Ascidien und Wirbelthieren. Arch. 
f. mikr. Anat., Bd. VI. pp. 115-172, Taf. VILL=-X. 


Korschelt, E., und Heider, K. 
93. Lehrbuch der vergleichenden Entwicklungsgeschichte der wirbellosen 
Thiere, Heft IIL. Jena. 


Kowalevsky, A. 
‘66. Entwicklungsgeschichte der einfachen Ascidien. Mém, Acad. St. Péters- 


bourg (sér. 7), Tom, X. 


Kowalevsky, A. 
‘71. Weitere Studien tiber die Entwicklung der einfachen Ascidien. Arch. f. 


mikr. Anat, Bd. VIT. pp. 101-180, Taf. XXII. 


Kowalevsky, A. 
‘92. Kinige Beitrage zur Bildung des Mantels der Ascidien. Mém. Acad. St. 
Pétersbourg (sér. 7), Tom. XXXVI. 


Lillie, F. R. 
95. The Embryology of the Unionide. Journ. Morph., Vol. X. pp. 1-100, 
Pl. I-VI. 


Loeb, J. 
‘91. Untersuchungen zur physiologischen Morphologie der Thiere. I. Hete- 
romorphose. Wiirzburg. [1890.] 


Loeb, J. ' 
"92. Untersuchungen, ete. II. Organbildung und Wachsthum. Wirzburg. 


82 pp., 2 Taf. 


Lwoff, B. 

‘94. Die Bildung der primaren Keimblatter und die Entstehung der Chorda 
wad des Mesoderms bei den Wirbelthieren. Bull. Société impériale des 
Naturalistes de Moscou. 1894, pp. 57-137, 160-256, 6 Taf. Also separate, 
177 pp., 6 Taf. Moskau. 


Mead, A. D. 
95. Some Observations on Maturation and Fecundation in Chetopterus per- 
gamentaceus, Cuvier. Journ. Morph., Vol. X. pp. 313-317, 1 Pl. 


Rabl, C. 
‘89. Theorie des Mesoderms. Morph. Jahrb., Bd. XV. pp. 113-252. 


Roule, L. 
‘84. Recherches sur les Ascidies simples des cdtes de Provence (Phallusiadées). 
Annales du Musée d’Hist. Nat. de Marseille (Zool.), Tom. IL, Mém. No. 1, 


270 pp., 18 Pl. 


CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 279 


Samassa, P. 
94. Zur Kenntniss der Furchung bei den Ascidien. Arch. £. mikr, Anat., 
Bd. XLIV. pp. 1-14, Taf. 1, LL. 


Seeliger, O. ; 
85. Die Entwicklungsgeschichte der socialen Ascidien. Jena, Zeitechr., Bd. 
XVIIL pp. 45-120, Taf. 1.-VIL. 


Stimpson, W. 
52. Several new Ascidians from the Coast of the United States. Proce. Bost, 
Soc. Nat. Hist., Vol. IV. pp. 228-232. 


Vejdovsky, F. 
88. Entwicklungsgeschichtliche Untersuchungen, Heft 1. Reifung, Befruch- 
tung und Furchung des Rhynchelmiscics. 166 pp., 10 Taf. Prag. 


Verrill, A. E. 
71. Descriptions of some imperfectly known and new Ascidians from New 
England. Amer. Journ. Sei. and Arts (ser. 4), Vol. L. pp. 93-100. 
Wheeler, W. M. 
95. The Behavior of the Centrosomes in the fertilized Egg of Myzstoma 
glabrum, Leuckart. Journ. Morph., Vol. X. pp. 505-411, 10 Figs. 


Willey, A. 
93. Studies on the Protochordata, 1. Cart. Journ. Micr. Sci., Vol. AXXIV. 
pp- 317-360, Pl. XXX., XXXL 


Willey, A. 
94. Amplioxus and the Ancestry of the Vertebrates. S16 pp. New York. 


Wilson, E. B. 
93. Amphioxus and the Mosaic Theory of Development. Journ. Morph. 
Vol VIIL pp. 579-638, 10 Plates. 


Wilson, E. B., and Mathews, A. P. 
95. Maturation, Fertilization, and Polarity in the Echinoderm Vgg. New 
Light on the “ Quadrille of the Centers.” Journ. Morph, Vol. X. pp. 619- 
342, 7 Figs. 


280 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 


EXPLANATION OF PLATES. 


All Figures were drawn with the aid of the Abbé camera lucida. The magnifi- 
cation is stated for each plate separately. ‘The egg membranes have not been rep- 
resented in any of the Figures. Arrows in the Figures connect sister cells, i. e. cells 
which have arisen by division of a common mother cell. For an explanation of the 
system of nomenclature employed, see page 226. 

In many of the Figures, only the cells of one half of the embryo (usually the 
right, that is, quadrants A and LD) have been lettered. The reader will be able 
readily to supply the deficiencies for the cells of the other half, since they are 
almost perfectly symmetrical in position with those that are lettered. Correspond- 
ing to each cell on one side of the median plane (in quadrant A or J) will be found 
a cell similarly situated on the other side of the median plane (in quadrant B or C); 
this cell should receive the same exponents as its mate, and either the letter B or C 
according as that mate is lettered A or D. Thus, the mate of A‘? in Figure 58 
(Plate X.) is the cell immediately to the right of it, which would be called 57.?; 
the respective mates of D*-* and D7-6 in Figure 57 would be called C7-5 and C™-5, 

In Figure 62 (Plate X.) and all following Figures, the endoderm cells are indi- 
cated by granular nuclei in a cell body that is left without tint or stippling; the 
chorda cells without tint or stippling, and with the outlines only of nuclei; the 
mesenchyme cells by a flat tint; the ring of neuro-muscular cells by stippling of 
the body of the cell. The ectoderm cells, since they are easily distinguishable 
from the endoderm cells, have been left, like the latter, without stippling or tint; 
their nuclei are sometimes drawn true to nature, i. e. with granulations, sometimes 
in outline only. 


ABBREVIATIONS. 
ast. 9 = female astral sphere. ms’chy. = mesenchyme. 
ast. 3 = male astral sphere. mu. = musculature. 
ed. = chorda. n. = nervous system. 
cl. pol. = polar globule. prnl. = pronucleus. 
cl. pol!- = first polar globule. prnl.2 = female pronucleus. 
ecdrm. = ectoderm. prnl.& = male pronucleus. 


en’drm. = endoderm. 


CastLeE. — Ciona intestinalis. 


PLATE I. 


All Figures represent sections; magnification, 560 diameters. Yolk granules are 
not represented, except in Fig. 2. 


Fig. 1. First maturation spindle, and formation of first polar globule. 
Fig. 2. Portion of section through recently impregnated egg. The spermatozoon 
lies at the centre of a region free from yolk granules. 

Fig. 3. Second maturation spindle. 

Fig. 4. Section through the same egg in region of male pronucleus. 

Fig. 5. Impregnated egg; male and female pronuclei visible in the same section. 

Fig. 6. Conjugation of pronuclei, viewed in the direction of the axis of the first 
cleavage spindle, which is in process of formation. (Cf. Plate IIL. 
Fig. 13.) 


GASTLE- GCIONA. 


Pra 


B.Meisel lith.Boston. 


CasTLe. — Ciona intestinalis. 


PLATE II. 


Figs. 7-10. Four sections through an impregnated egg ; magnification, 560 diam- 
eters. \ 

Fig. 7. Fourth section of series, showing the male pronucleus and archoplasmic 
spheres. Compare Figs. 11 and 12. 

Fig. 8. Seventh section of series, showing the female pronucleus. 

Fig. 9. Tenth section of series, showing the female archoplasm. 

Fig. 10. Twelfth section of series, showing polar globules. 

Fig. 11. Graphic reconstruction of the series on a plane, perpendicular to that of 
Fig. 7, indicated by the line ab, Fig. 7. 

Fig. 12. A similar reconstruction on a perpendicular plane, the projection of 
which is the line a’ b’, Fig. 7. 


CASTLE- GIONA. teks. Tl 


print. S 


LT'l.g 


WEG del. 


B, Meisel ith Boston. 


CaAsTLE, — Ciona intestinalis. 


Fig. 18. 


Fig. 14. 
Fig 15. 


Fig. 16. 


Fig. 17. 


Fig. 18. 


PLATE III. 


All Figures represent sections ; magnification, 560 diameters. 


Conjugation of pronuclei, viewed in the direction of the equator of the 
first cleavage spindle. (Cf. Plate I. Fig. 6.) 

First cleavage spindle. 

First cleavage nearly completed. Each of the newly formed nuclei is 
made up of two vesicles as yet incompletely fused. 

Section through one of the cells of a 2-cell stage, parallel to median plane 
of the embryo. 

Section from the same series as Fig. 16 through one of the two cells, near 
the median plane of the embryo. z, finely granular protoplasm, which 
marks the posterior-ventral side of embryo (cf. Plate VIII. Fig. 45, x), 
and is traceab!e up to the larval stage. 

Section through an 8-cell stage parallel to median plane of the embryo. 


GASTLE- GIONA. Piel: 


) 
. 
: 


WEG del. B Meisel, ith. Boston. 


a 


- ) “ 


CasTLe, — Ciona intestinalis. 


PLATE IV. 


Eight successive views of a living egg, seen from the left side; magnification, 3165 
diameters. Arrows indicate the direction of spindles. 


Fig. 19. Matured but unsegmented egg. 
Fig. 20. 2-cell stage. 

Fig. 21, 4-cell stage approaching. 

Fig. 22. 4-cell stage, “resting ” condition. 
Fig. 23. 8-cell stage, just formed. 

Fig. 24. 8-cell stage, nine minutes later. 
Fig. 25. 16-cell stage, just formed. 

Fig. 26. 16-cell stage, some minutes later. 


GASTLE- GIONA. 


Pie 


WEC del. 


B. Meise! th Boston. 


CasTLe. — Ciona intestinalis, 


PLATE V. 


Six successive views of a living egg, seen from the anterior end; magnification, 
315 diameters. Figs. 27-31 show the egg viewed as a transparent object. 
Fig. 32 is a surface view. 


Fig. 27. 2-cell stage, newly formed. 

Fig. 28. 2-cell stage, a few minutes later. 
Fig. 29. 2-cell stage, a few minutes later still. 
Fig. 30. 4-cell stage. 

Fig. 31. 8-cell stage, just formed. 

Fig. 52. 8-cell stage, a few minutes later. 


GASTLE- CIONA. : PES. 


oy CL.pol. 


WEC del. : B.Meisel ith Boston, 


CAsTLeE. — Ciona intestinalis. 


PGA V1: 


Magnification of all Figures, 315 diameters. Arrows indicate the direction of 
spindles. 


Fig. 33. Later stage of the same egg as that shown in Fig. 32, Plate V. 16-cell 
stage, just formed. 

Fig. 34. The same egg, passing into 24-cell stage. 

Figs. 35-38. Ventral aspect of four successive stages of a living egg. 

Fig. 35. 4cell stage. - 

Fig. 36. 8-cell stage, just formed. 

Fig. 37. 8-cell stage, a few minutes later. 

Fig. 88. 12-cell stage, just formed. 


CasTLE, — Ciona intestinalis. 


PLATE VII. 


Magnification of all Figures, except 43 and 44, 515 diameters; magnification of 


Figs. 45 and 44, about 500 diameters. 


Figs. 39-42. Four later views of the egg shown in Figs. 35-38, Plate VI.; same 


(ventral) aspect. 

16-cell stage, just formed. This view five minutes later than that in 
Fig. 38. 

16-cell stage, five minutes later. 

16-cell stage, ten minutes later still. Arrows indicate the direction of 
spindles. 

24-cell stage, just formed, some minutes later than the last view. 

A late 24-cell stage, viewed from the right side. 

Optical section of the same egg, near the median plane. The cells of the 
dorsal hemisphere are in mitosis. 


CasTLe. — Ciona intestinalis. 


PLATE VIII. 


Six successive views (obliquely from the left, above, and behind) of a living egg; 
magnification, 315 diameters. Arrows indicate the direction of spindles. 


Fig. 45. 2-cell stage. x, region of fimely granular protoplasm (cf. description of 
Plate III. Fig. 17). 

Fig. 46. 4-cell stage, just formed. 

Fig. 47. 8-cell stage, approaching. 

Fig. 48. 8-cell stage, fully formed (viewed as a transparent object). 

Fig. 49. 16-cell stage. ' 

Fig. 50. 24cell stage. During the formation of the 24-cell stage, the egg has 
rotated so that the view is almost exactly dorsal. 


CASTLE- GIONA. 


Pu. VIL 


B. Meisel ith Boston 


. 
: ora a. - 


CasTLe. — Ciona intestinalis. 


Fig. 51. 
Fig. 62. 


Fig. 53. 
Fig. 54. 


Fig. 55. 


Fig. 56. 


PLATE IX. 


Surface views of preparations; magnification, 400 diameters. 


24-cell stage, ventral view. 

The same egg, in the same stage, dorsal view (cf. Plate VII. Figs. 43 
and 44). 

32-cell stage, ventral view. 

The same egg and stage, dorsal view. 

46-cell stage, ventral view. 

The same egg and stage, dorsal view. 


CASTLE, — Ciona intestinalis, 


PLATE X. 


Figs. 57-62. Surface views of preparations ; magnification, 560 diameters. | 
Fig. 57. 48-cell stage, viewed from behind. | 
Fig. 58. The same egg and stage, viewed from in front. 
Fig. 59. 64-cell stage, ventral view. 7 
Fig. 60. The same egg and stage, dorsal view. 
Fig. 61. 76-cell stage, ventral view. | 
Fig. 62. The same egg and stage, dorsal view. 
Note. —Consult general statement under Explanation of Plates. The cell D*-§ 
and its mate in the left half of the Figure were stippled by mistake. 
Figs. 63-70. Eight cross-sections from a series through an embryo in late 76-cell _ 
stage; magnification, 560 diameters. For position of sections in em- 
bryo, see horizontal lines 63-70 in Fig. 62. 
Note. —In Fig. 63, the cells D7-6 and D*-14 should be stippled like their mates. 
In Fig. 67, the cell C71 should be e711, 


CASTLE. — Ciona intestinalis. 


PLATE XI. 


Magnification of all Figures, 560 diameters. 


Fig. 71. 112-cell stage, dorsal view. 

Fig. 72. Early gastrula, dorsal view. 

Figs. 73-77. Five cross sections through an early gastrula (128-cell stage). For 
position of sections in embryo, see horizontal lines 73-77 in Fig. 72. 

Note. — Consult general statement regarding lettering under Explanation of Plates. 

Fig. 78. Sagittal section through an early gastrula (older than that shown in 
Fig. 72). 

Fig. 79. Similar section through a slightly older stage. 

Fig. 80. Surface (dorsal) view of late gastrula. 

Figs. 81-83. Three horizontal sections from a series through a late gastrula. 


4 


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CasTLE. — Ciona intestinalis. 


PLATE XII. 


Magnification of all Figures, 560 diameters. 


Figs. 84-87. Four cross sections from a series through a gastrula with wide-open 
blastopore. 

Fig. 84. Section posterior to the blastopore. 

Fig. 85. Section through the posterior portion of the blastopore. 

Fig. 86. Section through the anterior portion of the blastopore. 

Fig. 87. Section anterior to the blastopore. 

Figs. 88-92. Five cross-sections through an embryo with greatly contracted blasto- 
pore; Figs. 88 and 89 posterior to blastopore ; Figs. 91 and 92 an- 
terior to blastopore. 

Note. —The large unstippled cell in the left half of Fig. 88 should have been 
stippled. 

Figs. 93-97. Five cross sections through aslightly older embryo. Compare vertical 
lines 93-97 in Fig. 98. Fig. 93, section posterior to blastopore ; 
Figs. 95-97, sections anterior to blastopore. 

Note.— The large unstippled cell in the left half of Fig. 93 should have been 
stippled, likewise two large cells situated laterally in Fig. 95, one in either half 
of the Figure. 

Fig. 98. Sagittal section through an embryo with nearly closed blastopore. 


CASTLE. — Ciona intestinalis, 


PLATE XIII. 


Magnification of all Figures, 560 diameters. 


Figs. 99-105. Seven sections from a series through an embryo with completely 
closed blastopore. Figs. 99-101, posterior to region of final closure 
of blastopore; Figs. 105-105, anterior to region of closure of 
blastopore. 

Fig. 106. Cross section through an early larval stage (unhatched). The trunk 


region is shown in the left portion of the Figure, the tail region in 
the right portion. 


Mar” 


Shas sss ie spsasesersee see ete 


The following Publications of the Museum of Comparative Zodlogy 
are in preparation : — 


Reports on the Results of Dredging Operations in 1877, 1878, 1879, and 1880, in Charge of ALEX- 

ANDER AGASSIZ, by the U. S. Coast Survey Steamer “ Blake,’ as follows: — 

A. MILNE-EDWARDS. Crustacea of the “ Blake.”’ 

E. EHLERS. The Annelids of the “ Blake.” 

G. B. GOODE and 'l. BEAN. Deep-Sea Fishes of the East Coast of the United States. 
* Blake ”’ and ‘‘Albatross’”’ Collections published in connection with the National Museum. 

A. A. HUBRECHT. The Nemerteans. 

C. HARTLAUB. The Comatule of the “ Blake,” with 15 Plates. 

A. E. VERRILL. The Alcyonaria of the “ Blake.” 


Illustrations of North American MARINE INVERTEBRATES, from Drawings by BuRK- 
HARDT, SONREL, and A. AGASSIZ, prepared under the Direction of L. AGASs1z. 


Selections from EMBRYOLOGICAL MONOGRAPHS, compiled by A. AGAssiz, W. Faxon, 
and E. L. MARK (discontinued for the present). 


A. AGASSIZ. ‘The Acalephs of the East Coast of the United States. 

Be On Dactylometra quinquecirra Agass. 
AGASSIZ and WHITMAN. Pelagic Fishes. Part II., with 14 Plates. 
LOUIS CABOT. Immature State of the Odonata, Part IV. 
E. L. MARK. Studies on Lepidosteus, continued. 

“ On Arachnactis. 
J.D. WHITNEY. Origin and Mode of Occurrence of Iron and its Ores. 

sé Nomenclature and Classitication of Ore Deposits. 
CHARLES WACHSMUTH and FRANK SPRINGER. ‘The North American Fossil 
Crinoidea Camerata. With an Atlas of 83 Plates. 


Contributions from the ZOOLOGICAL LABORATORY, in charge of Professor E. L, MARK, 
as follows: — 
W. WHITNEY. ‘The Histology of Thyone. 
T. G. LEE. The Suprarenals in Amphibia. 
Contributions from the GEOLOGICAL LABORATORY, in charge of Professor N. S. SHALER. 


Contributions from the PETROGRAPHICAL LABORATORY, in charge of Professor J. 
Evior WOLFF. 
Studies from the NEWPORT MARINE LABORATORY, communicated by ALEXANDER 
AGASSIZ. 
A. AGASSIZ and W. McM. WOODWORTH. Some Variations in the Genus Eucope. 


Reports on the Results of the Expedition of 1891 of the U. S. Fish Commission Steamer 
‘‘ Albatross,” Lieutenant Commander Z. L. TANNER, U.S. N., Commanding, in charge of 
ALEXANDER AGASSIZ, as follows: — 


A. AGASSIZ. The Pelagic Fauna. R. VON LENDENFELD. The Phospho- 
we The Echini. rescent Organs of Fishes. 
“ The Panamic Deep-Sea Fauna. C. F. LUTKEN. The Ophiurida. 
J. E. BENEDICT. The Annelids, O. MAAS. The Acalephs,. 
K, BRANDT. The Sagittz. E. L. MARK. The Actinarians. 
AS The Thalassicolz. G. W. MULLER. The Ostracods. 
C. CHUN. The Siphonophores. JOHN MURRAY. The Bottom Specimens. 
is T) - Eyes of Deep-Sea Crustacea. ROBERT RIDGWAY. The Alcoholic Birds. 
W.H. DALL. The Mollusks. P. SCHIEMENZ. Pteropods and Heteropods. 
C. B. DAVENPORT. The Bryozoa. W. PERCY SLADEN. The Starfishes. 
S. GARMAN. The Fishes. L. STEJNEGER. The Reptiles. 
A. GOES. The Foraminifera. THEO. STUDER. The Alcyonarians. 
H. J. HANSEN. The Cirripedsand Isopods. M. P. A. TRAUTSTEDT. The Salpidz and 
W. A. HERDMAN. The Ascidians. Doliolide. 
S. J. HICKSON. The Antipathids. E. P. VAN DUZEE. The Halobatide. 
W. E. HOYLE. The Cephalopods. H. B. WARD. The Sipunculids: 
G. VON KOCH. The Deep-Sea Corals. H. V. WILSON. The Sponges. 
C. A. KOFOID. Solenogaster. W. McM. WOODWORTH. The Planarians. 


Application for the Publications of the Museum should be made to the Director of the Museum 


of Comparative Zodlogy, Cambridge, Mass. 
ALEXANDER AGASSIZ. 


4 
es 
Ss 


PUBLICATIONS 
OF THE 


MUSEUM OF COMPARATIVE ZOOLOGY 


AT HARVARD COLLEGE. 


There have been published of the BuLtetrns Vols. I. to XXVI.; 
of the Memoirs, Vols. I. to XVIII. 

Vols. XXVIIL and XXIX. of the BuLietin, and Vols. XI. and 
XIX. of the Memorrs, are now in course of publication. 

The BuLietin and Memoirs are devoted to the publication of 
original work by the Professors and Assistants of the Museum, of 
investigations carried on by students and others in the different 
Laboratories of Natural History, and of work by specialists based 
upon the Museum Collections. 


The following publications are in preparation : — 


Reports on the Results of Dredging Operations from 1877 to 1880, in charge of 
Alexander Agassiz, by the U. S. Coast Survey Steamer “ Blake,” Lieut. 
Commander C., D. Sigsbee, U. 8. N., and Commander J. R. Bartlett, U.S.N., 
Commanding. 

Reports on the Results of the Expedition of 1891 of the U.S. Fish Commission 
Steamer “ Albatross,” Lieut. Commander Z. L. Tanner, U. S. N., Com- 
manding, in charge of Alexander Agassiz. 

Contributions from the Zodlogical Laboratory, in charge of Professor E. L. 
Mark. 

Contributions from the Geological Laborer, in charge of Professor N. S. 
Shaler. 

Contributions from the Petrographical Laboratory, in charge of Professor 
J. Eliot Wolff. 

Studies from the Newport Marine Laboratory, communicated by Alexander 
Agassiz. ; 


Subscriptions for the publications of the Museum will be received 
on the following terms : — 


For the Buttetin, $4.00 per volume, payable in advance. 
For the Memorrs, $8.00 ey es be 
These publications are issued in numbers at irregular inter- 
us;-one volume of the Bulletin (8vo) and half a volume of the 
temoirs (4to) usually appear annually. Each number of the Bul- 
letin and of the Memoirs is also sold separately. A price list of 
the publications of the Museum will be sent on application to the 
Director of the Museum of Comparative Zoélogy, Cambridge, Mass. 


ALEXANDER AGASSIZ, Director. 


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