Budding in Perophora ..

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BUDD^llii IN PEROPHORA

DISSERT AT I OK
Presented to the Board of University Studies of tne
Johns Hopkins University
for the
Degree of Doctor of Philosophy,
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buddtnct in pekophora

George Lefevre ,

Although the phenomenon of budding in the group of
Tunicata has received the attention of numerous observers for
a long time, during the past twelve or fifteen years investi-
gation has been largely confined to the study of the process
in the pelagic forms, Salpa, DoliolMm, and Pyrosoma . In the
Ascidians, hov/ever, our knowledge of the budding, until re-
cently, has been based almost entirely on the observations
of earlier workers. Among the latter, Kowalewsky, (12 and
IS) who studied the bud-development in Perophora, Didemnum,
and Amaroucium, Delia Valle (4) in Didemnum, Distaplia , and
Botryllus, and finally Seeliger (29) in Glavelina are espe-
cially prominent.

Within the last two or three years, hov/ever, our
knowledge has been greatly enriched by the researches of Pi-
zon (22) and Hjort (8) on Botryllus, and of Salensky (27) on
Distaplia.

Although the observations of these investigators

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agree on many important points, still on others, notably the
origin of the nervous system, they stand at the greatest va-
riance.

While the llarine Laboratory of the Johns Hopkins
University was stationed at Beaufort, N. C, during the sum-
mer of 1H94, I collected material for the purpose of studying
the development of the buds of Perophora viridis, Verrill,
which was found there in great abundance. This material I
supplemented the follov/ing summer at V/oods Holl, Mass. while
working in the laboratory of the United States Pish Commis-
sion.

My main object in undertaking the investigation was
to compare the bud -development of Perophora with that of Bo-
tryllus, as described by Hjort (8), and especially to deter-
mine, if possible, the origin of the nervous system.

The material, which was easily obtained at both
places in large quantities, proved to be most excellent for
my purpose, as it contained an unlimited supply of buds in
all stages of development.

The preserving fluids used were, 1, glacial acetic
acid; 2, a mixture of eighty parts concentrated corrosive
sublimate solution and twenty parts glacial acetic acid; and
3, Perenyi's fluid. The latter reagent gave perhaps the
most satisfactory results, although very good preparations
were obtained with the sublimate-acetic mixture, when the ob-
jects were left in it not longer than ten minutes.

Mayer's Haemalum proved to be most serviceable.

while borax carmine gave an excellent stain when used after
acetic acid or sublimate-acetic, but was of no value for ob-
jects fixed in Perenyi's fluid.

I have made use of Patten's method of orientation
(21) to great advantage, and have found it of invaluable as-
sistance in manipulating very small buds, which I was enabled
to cut with accuracy in any plane desired.

In studying the bud-development of Perophora I have
employed serial sections of all the stages in the sagittal,
frontal and transverse planes, and also a complete series,
throughout the entire development, of buds mounted in various
positions as total preparations.

The sections were cut from 3 to S/*- in thickness on
a Thome microtome, and all the drawings were made with the
aid of a Zeiss camera lucida.

I take much pleasure in acknowledging my indebted-
ness to Prof. Brooks, for the kindly interest with which he
has follov/ed my work, and for valiiable assistance given me.
I also desire to express in this place my great appreciation
of the many courtesies extended to me by the late Colonel
Marshall MacDonald at the Station of the 'J. S. Pish Gommis-

A preliminary account of some of my results '.vas
published in the Johns Hopkins University Circulars, No. 119,
June, 1895.

PEROPHORA VIRIDIS, VERRILI,.

This Ascidian, which is the only species of Pero-
phora known to occur on the Atlantic coast of North America,
was first found in Vineyard Sound by Vcrrill (34), and des-
cribed by him in 1871. A nev/ species. P.annectens, has re-
cently been reported from the coast of California by Ritter,
(23)

Perophora viridis grov/s luxuriantly below lov^-water
mark on the wharf piles in Little Harbor, V'oods Holl, and
Vineyard Haven, :iartha's Vineyard. I also found it to be
equally abundant during the summer months in the harbor of
Beaufort, N. C. The colonies form large thick clusters of
a beautiful greenish-yellow color, and usually occur together
with other Ascidians, and with Hydroids, Bryozca, Sponges
and Barnacles, the delicate stolons creeping over and cover-
ing the surfaces of everything within reach.

THE RUDIMENT OF THE BUD.

Budding in Perophora was first studied by Kowalew-
sky (12), v/hose careful work on this form laid the foundation
for all subsequent investigation of the process of budding
in the Ascidians .

■■ietschnikoff (18) had already discovered in Bo try 1-

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lus, that the bud-rudiment consists of two vesicles, one with-
in the other, the outer being derived from the ectoderm, and
the inner fron the peribranchial wall of the parent. He al-
so observed the splitting up of the inner vesicle to form the
median branchial sac and the lateral peribranchial spaces,
but neither Metschnikoff nor Krohn (15) who confirmed these
observations on the Botryllus bud, sav/ at all clearly the de-
tails of the development. This was left for Kov/alewsky, who
described many of the internal processes occurring in the
bud-development of Perophora Lister i . He show^ed that in Pe-
rophora also the young bud is composed of two vesicles, the
outer one being derived from tr.e ectodem and the inner from
the partition wall of the stolon. According to Kowalewsky's
account the endodermal or inner vesicle becomes divided com-
pletely into three portions, the two lateral fusing dorsally
and form.ing the peribranchial cavity, and the median giving
rise to the branchial sac. I shall try to show below that
in Perophora viridls, at all events, the peribranchial cavity
is formed by quite a different process.

The origin of the bud as a double-v.'alled vesicle
has been verified by all subsequent investigators, and tho-
roughly established as a type of development cormion to all
Ascidians whose budding has been studied.

The outer wall of tne vesicle is directly derived
from the ectodem of the parent animal, and becomes the ecto-
dermal covering of the bud. According to the majority of
investigators this outer layer takes no active part in tiie
furtner development, but Salen,3ky (27) and Oka (20), as v;ill

be pointed out belov>f, inaijitain that the ectodem is concerned
in the formation of the nervous system.

As to the derivation of the inner wall, of t.ie ves-
icle, the case is not so simple, for in different Ascilians
this layer arises from entirely different parts of the parent,
coming in some forms from an endodermal, in o triers from an
ectodermal structure. In Perophora and Glavelina it is de-
rived from the parti t ion-v;all of the stolon, whicn in the
latter, and presumably in the former, is of endodermal origin;
in Didernnurn and Distaplia from the wall of the gut, and in
the Polyclinidae from the endodermal wall of the post-abdomen.
In all the above mentioned species, then, th« inner ves-
icle of the bud-rudiment is derived from an endodermal struc-
ture. In Botryllus , however, this inner vescicle is fonned
directly as an evagination of the outer v^all of the peribran-
chial space, whose origin in the Ascidian larva has been a
question of much dispute. Kowalewsky (11) first showed that
in the larva of simple Ascidians the peribranchial cavity is
formed from two symmetrical ectodermal invaginations, which
later fuse together dorsally and surround the branchial sac.
Delia Valle (4), however, contradicted these results, and
maintained an endodennal origin for the peribranchial sacs in
both simple and compound Ascidians, and Pizon (22) has re-
cently described the sacs as arising from two divertecula
from the branchial sac in the larva of Botryllus. Kowalev/-
sky's account is borne out by all the later investigation of
the subject, with the exception of that of Pizon. Seeliger

(29: Clavelina) , 7Jilley (35: Ciena) , Salen.sky (28: Diploso-
aa, Didemn-on) , and Caullery (1: Dlstaplla,Tjeptoellnum, Qlos-
sophorum, CircinaliujTi) , agree in their statements that the
peribranchial sacs arise in t"ne manner described by Kowalew-
sky, and are therefore purely ectodermal. It is fairly safe
to assume that Pizon is v.frong, and tnat in Botryllus, as in
all other Ascidians studied, the sacs are ectodermal. Tf
triis be the case, as the inner vesicle of the bud-rudiment
is directly cut off from the outer peribranchial wall in both
larva and buds, it follov;s that all the organs, including the
peribranchial sacs, derived from this inner vesicle through-
out the entire series of buds proceeding from the larva, are
ultimately of ectodermal origin.

After this brief review of tne state of our knov;-
ledge concerning trie origin of tne bud-rudiment in the Asci-
dians, I shall now describe the process, as I have found it
to occur in Perophora viridis .

The buds arise in a single rov/ along the course of
the delicate stolons, which branch profusely and adhere on
one side to the surface over which they creep. The branches
come off approximately at right angles to the stems, from
which they proceed, but aside from this char;icteris tic there
is no regularity whatever in the method of branching; they
arise at unequal intervals, and as each grows out, it gives
off in its turn still younger shoots, the whole system be-
coming much tangled and twisted to form a loose felt-work.

Proceeding towards the growing tips of the stolons,
both buds and branches are seen to become successively young-

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er .

In Fig. 6, Is sho-^n a transverse section of a sto-
lon. The outer wall of trie hollow cylindrical tube consists
of a flattened epitheliini, the ectoderm, ^ct., covered exter-
nally by the transparent cellulose test, _t. Running from
one side to the other the partition-wall, p_. t . , is seen in
cross-section, completely dividing the tube into two com-
partments, in which the free cells of the blood lie scattered
about. The stolonic partition consists of a double lamella
made up of flat, attenuated cells, and may be described as a
collapsed cylinder, the walls of which are closely pressed
together, and attached along the upper and lower borders to
the inner surface of the ectodermal tube. It divides the
stolon longitudinally into halves, and stretches entirely
across the lumen, although in preserved material it is usual-
ly more or less shrunken away from the outer wall. The two
compartments of the stolon are in open communication with the
body cavities of the animals, so that a free circulation of
blood occurs from one individual to another.

As Kov;alev;sky (12) has pointed out, the partition-
wall ( "Scheidewand ") does not reach quite to the extreme dis-
tal end of the stolon, but there the two spaces or sinuses
open into each other. The blood flows in opposite direction;
in the two sides of the stolon, up one to the tip, around the
free end of the partition, and down the otner. v/hen the
heart-beat is reversed, of course the direction of the blood-
flovf in tne stolon is changed.

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The buds always arise in the plane of the partition
wall, on the side of the stolon opposite to that which is at-
tached to the underlying surface; the line of attachment is,
therefore, parallel to and immediately beneath the lower bor-
der of tne partition. It will be seen further on that the
definitive s-agittal plane of the bud coincides with the plane
of the partition-wall, and hence the latter structure divides
the stolon into a right and left half in reference to the
parts of tne bud, and not into a dorsal and ventral portion,
as described by Kowalewsky (12), whose well-knov/n figure of
the P^erophora stolon is consequently misleading. Looking
at the lateral surface of the buds, as shown in that figure^,
the partition-wall would be seen from the flat side and not
on edge.

The first indication of tne appearance of a bud is
a slight bulging out of tne ectoderm of the stolon at one
point, and if a transverse section be taken at this spot, it
will be seen that the two layers of the partition-wall have
spread widely apart, giving the appearance of a more or less
spherical vesicle. This is well shown in Pig. 7. The
walls of the partition are seen to be no longer composed of
flat cells, except on the lov/er side, that is, the side to-
wards the surface of attachment, but have become very much
thickened by active cell-multiplication. The cells, toe, of
the ectoderm over the thickeiied portion of the partition have

-'■ This figure is reproduced in Korschelt and Heider's "Ent-
wicklungsgeschichte",page 1366 .

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increased in height and are now nearly cuboldal. The por-
tion, where it is s-^rollen out into a vesicle, becomes drawn
away from the outer wall of the stolon. I do not think
that this is due to shrinkage, for it is invariably found,
and moreover, beyond this region on ei trier side, where the
partition is still flat, it is seen to stretch clear across
the lumen of the stolon.

The ectoderm continues to bulge out more and more,
until it forms a hemispherical protuberance on the surface
of the stolon. The cells composing the raised portion of
the ectoderm do not remain cuboidal, but from now on, as the
swelling increases, become gradually flattened again. The
thickened portion of the partition keeps pace with the ecto-
dermal evagination, and grov/s out into the latter, but the
thin walls below nov; come together, and close off tne upper
portion as a thick-walled vesicle, without, however, severing
the connection. This process is readily understood from
Pig. 8, in which the walls of the lower part of the partition
have become united again.

In Pig. 1, a bud is shown at tnis stage from the
exterior.

As the ectoderm continues to push out, it becomes
constricted where it passes over into the stolonic ectoderm,
the constriction being greater at first before and behind
than on the sides. The bud-rudiment, which by this time is
almost spherical, is sharply marked off from the stolon, and
stands eitner straight out as a round knob from the surface

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of the latter, or is slightly inclined towards its free tip;
Pig. 2.

It novi represents the characteristic double vesicle
of the young Ascidian bud; the outer or ectodermal vesicle is
directly derived from the ectoderm of the stolon, and the
inner or entodermal vesicle, v/hich has become a closed sac,
arises by evagination of the thickened portion of the dilated
stolonic partition. The connection between the partition
and the inner vesicle is retained for a long time, and the
cavity of the latter is to be regarded as being in communi-
cation with the potential cavity of the partition.

A transverse section at this stage, Fig. 8, shows
the still somewhat thickened cells of the ectoderm of the bud
rudiment, and the endodermal vesicle, lying inside, v;-i th its
much thickened v/alls. Active cell-multiplication has been
going on in the latter, which are of uniform thickness, ex-
cept below where they pass off gradually into the thin walls
of the partition. The cells of the blood are found scatter-
ed about betv/een the two vesicles, and it is to be especially
noted that many are lying close against the inner surface of
the ectodenn and outer surface of the endoderm at niimerous
points .

The bud-rudiment does not long remain spherical,
but soon becomes elongated by a grovTth towards tne apex of
the stolon. It now assumes an oval shape, and one end lies
free over the surface of the stolon beyond the portion which
is directly connected with the latter, Pig. 3. It is the
free end which is later to be distinguished as the anterior

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portion of the bud, v/hile by this process of elongation an-
teriorly the connection with the stolon comes to be left be-
hind in the posterior region. The side next th^'e stolonic
wall becomes the ventral surface of the bud, and that oppo-
site it the dorsal.

The protuberances of the stolonic ectodenn, which
Seeliger (29) describes as occurring at the base of the bud-
rudiment in C_lavelina, are not present in Perophora ; they
are merely simple ectodennal sacs, filled with blood-cells,
and do not contain an evaginatlon of the partition-wall.
Seeliger calls them "TIahrkanrnern", and regards them as res-
ervoirs of food-material for the developing buds.

Before going on to describe the origin of the va-
rious internal organs, some of which have by this time begun
to make their appearance, I wish to say a word about the par-
tition-wall of the stolon.

A.S the inner vesicle of the bud is formed entirely
from this structure, it is a matter of importance to know
from what it is derived in the larva.

It has never been observed in Perophora how the
stolonic partition arises, although it is usually supposed
to be of endodermal origin from its likeness to the similar
structure in Clavelina.

Van Beneden and Julin (33) have shown that in the
larva of Clavelina it is formed as a direct continuation of
the epicardiim, which arises as a diverticulum from the pos-
terior wall of the branchial sac, and is therefore entirely

■12.

endodermal.. During the month of August, 1895, I made an
attempt to discover the ori;^in of the stolon in Perophora.
Larvae were put into aquaria, through which water was kept
constantly flowing, and although they settled down and under-
went tiie metamorphosis, at the expiration of nearly four
weeks, not the trace of a stolon sprouted from them. VJhen
larvae were afterwards sectioned and studied, nothing like
an epicardium, such as occurs in Clavelina, was found.

If then such a structure exists in the larvae of
Perophora, it must be formed at a very late period. As I
shall try to show further on, it is doubtful whether there is
a true epicardium. in the buds of this Ascidian, and it is
possible that it is also absent in the larvae.

The further Development of the Bud.

At the time when the bud-rudiment begins to e-
longate, or very shortly after, the rudiments of several nev/
structures are laid down. These are the (1) pericardium;
(2) the peribranchial sacs; (3) the d_orsal tube ; (4) the gut;
and (5) the ganglion. These do not all arise sim.ultaneously
and although the rudiment of the pericardium is the earliest
to appear, it will be necessary to describe the formation of
the peribranchial sacs first, in order to render intelligible
certain relations between these and other structures.

Origen and Development of _th_e Peribranchial Cavity.

All investigators agree in deriving the peribran-
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chial cavity from the inner vesicle of the bud-rudiment, but
the manner in which it arises is not the same in all species

of Ascidians.

e

In Perophora , Didennum, and Amarouium, according

to Kowalewsky (12 and 13) two parallel longitudinal furrows
appear on the outside of the inner vesicle, and by gradually
deepening finally divide the latter completely into three
portions. The two lateral divisions which are thus cut off,
grow up over the middle one, and fuse to form the median por-
tion of the peribranchial cavity, which now surrounds the
branchial sac dorsally and laterally.

Seeliger (29) has described a different method of
formation of the peribranchial cavity in Glavelina. Accor-
ding to him the inner vesicle is not divided into three por-
tions, but into two, one of which, the posterior, gives rise
to the branchial sac and the gut, while the other forms the
whole peribranchial cavity. These results were contradicted
by Van Beneden and Julin (33), who maintained that in Glave-
lina, the process is the same as that described by Kowalev/sky-

As Seeliger's view was not founded on an investigation of
an unterrupted series of stages, and as there were wide gaps
in his observations at periods which are especially concerned
in the formation of the peribranchial cavities, the supposi-
tion that his results are wrong is very probable. In Dis-
taplia Salensky (27) has shown, and his results have been
confiiined by Hjort and Prl . Bonnevie (10), that the inner
vesicle gives rise to two lateral evaginations , which become
completely constricted off as separate vesicles, the peri-

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branchial sacs, and gradually extending dorsally, fuse to-
gether on the median line. These sacs are not formed at the
same time, but one is given off from the inner vesicle before
the other, so that at a very early stage an asymmetry of the
bud is produced.

Delia Valle (4) described a sim.ilar method of form-
ation of the peribranchial cavity for Botryllus , but both
Pizon (22) and Hjort (8) have conclusively proven his obser-
vations to be erroneous.

According to these authors both the lateral and me-
dian portions of the peribranchial cavity arise at the same
time as a saddle-shaped bag, which is cut off by two longi-
tudinal furrov/s from a median vesicle, the later branchial
sac. Hjort regards this process as a great curtailing of
the embryonic development, such as often takes place in buds.

Salensky (27) confirms this conclusion, but goes a step
further, saying that, "die Entwicklung der Peribranchialhohl-
en des Botryllus eine Abkurzung nicht nur beziiglich der era-
bryonalen Entwicklung, sondern auch beziiglich der Entwicklung
dieser Organe in den Knospen anderer Ascidien darstellt."

My observations on the development of this struc-
ture in Perophora viridis, although agreeing with those of
Iijort, in so far as they show that the peribranchial sacs do

not arise separately as closed vesicles, which later unite to

the
form clcacal cavity, indicate that the process is not so sim-
ple as that which occurs in Botryllus .

If a transverse section of a bud be examined about

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the time when the elongation spoken of above is just begin-
ning, it will be found that the -vvall of the inner or endoder-
mal vesicle is no longer of uniform thickness. ?ig. 9, is
drawn from such a section. The ectoderm covering tne bud,
although it is not shown in the figure, has again become
flattened after its temporary thickening, and is nov/ like
that of the stolon. ^ The figure clearly shov/s that the sto-
lonic partition is made up of two lamellae which are contin-
uous below Y/ith each other and pass over above into the walls
of the inner vesicle. The important change to be noted, how-
ever, is that the wall of the endodermal vesicle on one side,
the left, is getting perceptibly thinner than elsewhere, and
that the whole vesicle is no longer symmetrically placed in
reference to the stolonic partition, but is bulging out
slightly to the right. This is the first indication of a
marked change -Thich is about to take place in the internal
relations of the bud-rudiment.

By a peculiar process, which may be described as a
transverse or rotatory growth affecting the inner vesicle, the
thicker wall of the right side is carried or pushed down
gradually until it comes to lie eventually on the ventral
side, that is, the side next to the stolon.

The stolonic partition remains stationary, and the
displacement or shifting around of the inner vesicle takes
place on this as a fixed support. The process might be il-
lustrated by the drooping of a flower to one side on its
stem, although the change of position cannot be a purely
passive falling over of the vesicle. In Fig. 9, a small

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collection of cells, p c . , is seen adhering to the -.vail of
the vesicle high up on the right side, which, as we shall
see below, is the rudiment of the pericardium. This cell-
mass remains fixed at the same place on the wall, and during
the shifting of the vesicle is borne dov/n towards the ventral
side, describing in its descent an arc of about 90 . It
therefore furnishes a good register of the pi'ogress of the
displacement of the vesicle.

As the turning proceeds, the difference in thick-
ness between what was at first nearly the whole right side,
and the rest of the vesicle, becomes more marked; conse-
quently the cells composing the entire vesicle, except in the
thicker region, are seen to be growing more and more flatten-
ed.

The displacement is most probably brought about by
a rapid growth and stretching of the cells composing the
greater portion of the vesicle, whereby the actual right side
which is morphologically the ventral side of the vesicle, is
shifted or pushed ventrally through 90*^ .

This process is analogous, at all events, with the
rotation or displacement of the pharynx of the Amphioxus lar-
va from, right to left, although I am not prepared to claim
any phylogenetic relation between the two.

By comparing Fig. 9, with Pigs. 10 and 11, the pro-
cess can be readily understood. The shifting involves the
anterior end of the vesicle only to a slight extent. In
this region a difference in thickness of the walls is not ob-

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served, and the rudiment of the dorsal tube, which defini-
tively has a median dorsal portion in the anterior end of the
bud, arises as a collection of cells, almost at the same time
that the pericardial rudiment appears, lying a little to the
left of the mid-dorsal line on the wall of the vesicle, d.t.
Fig. 28. If the displacement took place to as great an ex-
tent anteriorly as posteriorly, it is evident that this cell-
mass would appear much further down on the left side; but
that the anterior end is slightly rotated is shown by the
fact that the rudiment appears not exactly in the median line
where it is eventually brought through the shifting of the
vesicle, but somewhat to the left of it.

The formation of the peribranchial cavity is asso-
ciated with this change of position of the endodermal vesi-
cle. In Figs. 10 and 11, it is seen that the lov/er portion
of the vesicle at the point indicated by the line a_, is being
bent in, with the result that the wall in this region makes
two angles, one directed inward and the otner outward, a and
_b, Fig. 11. The apex of the latter marks a point on the
wall of the vesicle, which will have travelled through 90 ,
when the displacement is completed, as its final position
will be in the mid-ventral line.

As the inv/ardly directed fold deepens, it gradually
divides off a portion of the inner vesicle on tne left side,
which is connected with the stolonic partition; this is the
left peribranchial cavity, 1 .pbr . c . Pigs. 11 and 17. This
fold begins somewhat in front of the middle of the vesicle.

•18-

and deepenirig rapidly in this region, gradually extends pos-
teriorly.

As these changes are going on, the connection with
the stolonic partition is gradually becoming co: stricted, and
is now only present in the posterior half of the bud, while
at the same time the ectodermal stalk is also getting narrov/-
er. Ritter (24) in a preliminary note on the budding of Pe-
rophora, says that, "When the differentiation of tne 'endo-

derm' into the branchial and two peribranchial sacs, takes

tnat the de\/elopJng-
place, it does so in such a way blastozooid is connected v/ith

the double walled partition of the stolon, not by the bran-
chial sac, as has been hitherto supposed, but by the left pe-
ribranchial sac." He does not, however, describe how this
comes about. Prom an examination cf Pigs. 10 and 11, it is
readily understood. The communication of the body-cavity of
the bud with the blood spaces of the stolon is never complete-
ly closed, as there is always a free circulation of blood
from the one to the other, but eventually the left peribran-
chial cavity is entirely severed from the stolonic partition.

I cannot, hov/ever, confirm Ritter 's statement (I.e., page
367), that this connection is lost at an early stage, namely,
"at a time v/hen the two peribranchial pouches have merely be-
gun to envelop the branchial sac." I find that it persists
for a very much longer time, and is still present, although
greatly constricted, at a stage when some of the gill-slits
have been formed and the peribranchial cavity has been wholly
separated from the brancnial sac. Pig. 21, shov/s the con-

•19-

nection at such a stage.

The first indication of the right peribranchial
cavity is a slight longitudinal folding-in of the wall of the
inner vesicle, some distance up on the right side, v/hich ap-
pears after the shifting of the vesicle has begun. This
furrov/ starts a little in front of the anterior terrranation
of the left peribranchial fold, and as it deepens and extends
posteriorly, it is gradually carried down towards the ventral
side, in the same way as the pericardial rudiment. It is
already present at the stage represented in Fig. 11, but has
not yet reached back far enough to appear in a section which
shows the left fold. In Fig. 12, which is taken from the
same series of sections, but a little further forward, it
is v/ell marked, r . p hr.c .

As the shifting continues, the inner vesicle tends
more and more to assume a symmetrical position. The two
furrows, which deepen rapidly and run in obliquely to meet
each other, do not come together on the dorsal surface of the
vesicle, but some distance below it. The result of this is,
that when the right and left peribranchial cavities are sep-
arated from the inner vesicle, a median dorsal portion con-
necting them, is constricted off at the same time. This me-
dian piece, hence, does not arise, as Kov/alewsky (12 and 13)
describes, from the fusion of the lateral sacs dorsally, but
the three portions are cut off together. We now find a me-
dian vesicle, the later pharynx, surrounded dorsally and lat-
erally by a U-shaped bag, which consists of the dorsal or

-20-

cloacal and the lateral divisions of the peribranchial cavi-
ty. This is essentially the same process as that which Pi-
zon (22) and Hjort (8) have described for Botryllus .

In Perophora the folds which separate the peribran-
chial cavity from the inner vesicle, do not involve the en-
tire length of the latter, but leave nearly the whole of the
anterior half undivided, as well as a short region at the
posterior end of the vesicle. And further, the whole peri-
branchial cavity is not constricted off at the same tir.e, but
as stated above, the furrows begin anteriorly and extend
gradually back, so that at any given stage the opening of the
median vesicle into the peribranchial cavity is much wider in
a posterior section than in one further forward.

V^hen the right peribranchial cavities are being
separated off, as just described, a broad pouch or divesti-
culum grov/s out from the anterior margin of each, and by de-
grees spreads over the undivided portion of the inner vesi-
cle. These pouches are direct continuations of the lateral
Cavities, and later completely cover the sides of the ante-
rior region of the peribranchial sac, but they never fuse
dorsally.

Similar extensions are carried out from the poste-
rior margin of the lateral cavities, and though not promi-
nent at first, still, as the bud gets older and increases in
length, they attain a considerable size and surround a part
of the digestive tract.

The peribranchial cavity nov/ consists of two deep

-21-

lateral sacs, surrounding the spacious branchial sac, and
connected dorsally bj'^ a median space, the cloacal cavity.
The lateral sacs are unsymmetrical, however, until quite a
late stage, for the anterior pouch of the right peribranchi-
al sac grov/s more rapidly and extends further forward than
the sirrilar pouch on the left side, while the posterior pouch
of the left side extends further backv/ard than the corres-
ponding one on the right side. Eventually the two sacs be-
come symraetrical.

The formation of the peribranchial cavity is eas-
ily understood from the series of sections represented in
Pigs. 13--1S; these v/ill be rendered more intelligible by a
comparison with Pig. 4, which is drawn from a total prepara-
tion of a bud at the same stage of development. The sec-
tions are taken respectively at the levels indicated by the
parallel lines, a_, _b, _c, d, e^ and f_ of Pig. 4. In Pig. 13,
line a of Pig. 4, the most anterior one of the series, the
branchial sac, br, is seen by itself, for the extensions of
the peribranchial sacs have liot re.ucle-ct far enoi.'gii forv/tird to
appear in the section; the hypophyseal tube, d . t , is shoivn
on the dorsal side of the branchial sac. Fig. 14, line _b,
only includes the anterior extension of the right sac, for,
as just stated above, the pouch on the opposite side lags be-
hind, in its growth. Fig. 15, line c, represents a section
taken just in front of trie anterior face of of the cloacal
cavity, and shov/s both peribranchial sacs at the level where
they are continued foTtfard into their anterior extensions.

In Pig. 16, line d., trie section passes through tiie anterior
portion of the cloacal cavity, cl, which is seen to connect
the lateral sacs; the constriction, which will untirnately
completely cut off the U-shaped bag from the median vesicle
has proceeded in this region to a considerable extent, and
has greatly narrowed the opening between the peribranchial
and branchial cavities. In a section further back. Fig. 17,
line e, the folds which are foniing the peribranchial sacs,
are much less deep and v/ider apart; the connection between
the left sac and tne stolonic partition is present in this
region. Finally, Fig. IS, line _f, represents a section be-
yond the peribranchial sacs, the posterior pouches of which
at this stage have not yet begun to grow out; the section
passes behind the connection with the stolonic partition, but
through the storaach, s^t^, which is seen on the left side.

The further development of the peribranchial cavity
from this stage on merely consists in the completion of tne
constriction, whereby the 'J-shaped bag is completely cut off
from the branchial sac, and in the extension of tne anterior
and posterior pouches of the peribranchial sacs, which final-
ly surround the whole pharynx laterally. These relations
are illustrated by the series of sections. Figs. 19--21, which
are taken from the same bud. Fig. 19 is a section through
the anterior end of the bud, and shows tne lateral extensions
of the peribranchial sacs surrounding the pharynx. The peri-
branchial cavity is now entirely cut off, and its lateral
portions are united in the middle region of the bud by the

-23-

dorsal connecting piece or cloaca; this condition appears in
Pig, 20. Beyond the cloaca, the posterior pouches of the
lateral sacs, which like the anterior pouches are not united
dorsally, are seen in Fig. 21; the connection of the left
sac witn the stolonic partition in tnis figure has already
been referred to. By this time the process of displacement
is completed, and the definitive syminetrical arrangement of
the pharynx and peribranchial cavity is reached. The con-
nection between the left peribranchial sac and tae partition
of the stolon is nearly severed; it is found in only two
sections of this series, one of which is seen in Fig. 21.
A total preparation of a bud at about this stage is shown in
Pig. 5.

Epicardiam. This structure was first described
by Van Beneden and Julin (33) in the buds and larvae of
Clavelina, and was shown by these authors to be closely con-
nected with the development of the pericardimi. It arises
as an evagination of the posterior wall of the branchial sac,
and a little further back divides into two blind pouches,
which remain separate in the buds, but in the embryo unite to
form the "cul de sac epicardique" of Van Beneden and Julin;
the latter is continued into the stolon to form the double-
walled partition. The development of the epicardium will be
again referred to in connection with the pericardimn, with
which it stands in very close relation in some Ascidians.

In Distaplia Salensky (27) has described the epi-
cardial sacs as arising in the buds at an early stage by eva-

-24-

gination from the posterior end of the inner vesicle; the
two sacs are not formed at the same tine, and the left one
is always larger that the right.

In the buds of the Polyclinidae the epicardium is
formed in the same 7.'ay; two small diverticx^la, a right and
a left one, are given off from the posterior end of the bran-
chial sac, from which they afterv/ards become detached. They
soon, however, unite to form a single tube, which is contin-
ued out into the postabdomen, where it is destined to fur-
nish the inner vesicles of the buds produced by transverse
constriction of that region of the body.

The existence of an epicardium in Botryllus is de-
nied by Hjort (8), but maintained by Pizon (22). According
to the latter the inner vesicle at a very early stage gives
off two anterior lateral diverticula, one on each side, which
later form the peribranchial cavity, and also two posterior
lateral diverticula. These four pouches are at first sepa-
rate, but soon the two on each side fise in the middle region
of the bud. V^hen the peribranchial cavity is separated from
the inner vesicle, the posterior diverticula are cut off at
the sane time, and now appear as posterior prolongations of
the cloacal cavity, with which they always remain in free
communication. They are what Pizon calls the "diverticules
perivisceral^;:" , and in later stages completely envelop the
digestive tract. From the fact that these cavities arise
as two diverticula from the posterior end of the inner vesi-
cle Pizon regards them as homologous with the epicardial

-25-

tubes of other Ascidians, and states (I.e., page 29), that
"la formation de cette cavite peri viscerale n 'est pas secon-
daire ©t qu'elle s'est annoncee, des le debut, par deux pe-
tits diverticules posterieurs de la vesicule primitive, en
niemetenps que les diverticules anterieurs correspondants qui
engendreront la cavite peribranchiale . "

These perivisceral diverticula, however, differ
from the epicardial tubes of Clavelina, Distaplia and the
Polyclinidae in that they communicate with the cloacal cavi-
ty.

Hjort (I.e., page 594) states, that the "einheit-
liche Peribranchialblase sich nun derart weiter entwickelt,
das sie nicht nur den Abscnnitt des Kiemendarnies , sondern
den ganzen Darmtractus unwachst", and Salensky (27), who ac-
cepts the conclusion of Pizon as to the homology of the peri-
visceral diverticula, thinks that Hjort evidently saw the
'epicardial sacs' in Bo try 11 us, but failed to recognize them
as such. Salensky believes that the connection of the 'epi-
cardial sacs' with the cloaca in Botryllus, must be regarded
as a result of the early separation of the peribranchial ca-
vity from the inner vesicle.

In the light of these considerations it is possible
that the posterior extensions of the peribranchial sacs,
which I have described as arising in the buds of Perophora
viridis,are likev/ise homologous with the epicardial tubes of
other Ascidians. It is to be remembered, however, that if
such be the ease, which I think very doubtful, their direct

origin from the inner vesicle has been completely lost, as
they do not appear until quite a late stage, and then merely
as prolongations backi^^ard of the lateral portions of the pe-
ribranchial cavity, after the latter have been entirely cut
off from the inner vesicle. This would, therefore, be a
still more modified condition than that which is found in Bo-
tryllus .

Pizon (I.e., page 105) makes the statement, which
is not, however, illustrated by figures, that he has confirm-
ed on the buds of Perophora Listeri the results of Kowalewsky
(13: Amaroucium proliferum) and of Van Beneden and Julin (33:
Glavelina Rissoan-t) in regard to the origin of the epicardial
tube. "Ce tube", he says, "resulte bien de la reunion de
deux petits diverticules qui naissent a droite et a gauche
du sac branchial et qui s'isolent conpletement de celui-ci
a un moment donne . " Such a description is not in the slight
est accord with my obser-'ations, and if an epicardial tube
arises in this manner in the buds of the European Perophora,
it certainly does not in Perophora viridi s.

The Branchial Sac or Pharynx. That portion of the
original inner vesicle, which is left after the separation of
the peribranchial cavity, becomes the pharynx. At its ante-
rior end it finally opens to the exterior through the bran-
chial orifice, and after the appearance of the gill-slits it
comjnunicates with the peribranchial cavity, w..ile posteriorly
it leads off into the digestive tract.

-27-

The formation of the branchial sac in the buds of
Perophora viridis is complicated by reason of the peculiar
shifting of the inner vesicle, which has been described a-
bove . The whole vesicle, with tne exception of tne anterior
end, Y/hich, as already stated, is but slightly involved in
the process, becornes shifted or revolved through about 90 ,
in such a way that the original right wall of the vesicle
comes to lie ultimately on the ventral side. This right
wall, as has been shown, is early found to be much thicker
than the rpst of tne vesicle, the difference being due, not
to an increase in thickness of this region, but to the flat-
tening of the cells composing the remaining portion of tne
vesicle. It is this thickened v/all, originally on the right
side, which forms the floor of the pharynx in that part of
the vesicle which is concerned in the displacement. Very
soon after tne beginning of the change in position, a shallow
longitudinal groove is found on the inner surface of the ves-
icle in the middle region of the bud, lying on tne rignt side
on a level with the lov;er border of the pericardial rudiment.
This is the first appearance of the endos tyle ; its position
is shown at end in Fig. 10, but at a slightly earlier stage
it is found even higher up. In this figure and the next two
it is seen that tne groove runs through about the middle of
the thickened area, that is to say, above and belov; it there
are equal portions of tne thick wall, which will lie to its
right and left when the change in position of the vesicle is
fully accomplished.

• 28-

The groove rapidly extends anteriorly and posteri-
orly, and at the same time becomes deeper and broader. When
it reaches its definitive position in the mid-ventral line,
it stretches throughout the entire length of the branchial
sac.

It v/ill not be necessary to speak of the differen-
tiation of the endostyle into the various zones of cells
which go to make it up, as these have been described by nu-
merous authors: Delia Valle (3), Herdmann (7), Lahille (17),
and others.

It is to be especially noticed in Figs. 10, 11, 17
and 21, that the rositions of tne pericardial rudiment and
endostylar groove in reference to each other remain the same
during the displacement of tne vesicle. Prom this fact it
is evident that the thick portion of tiie vesicle is carried
down bodily, and that no interstitial growth takes place in
this region during the process, else the distance betv/een the
pericardium and endostyle would not remain the same. It
cannot be said that the pericardial rudiment might compensate
by its own growth for any increase in extent of that part of
the wall against which it lies, for it covers practically
about the same area as long as it adheres to the vesicle.
It would seem, therefore, that the change in position of the
inner vesicle is brought about by the stretching out and
flattening of the cells in all but the thick area, and that
the latter is borne or rather pushed dov/n tov/ards the ventral
side .

-29-

The Gill-Slits . The gill-slits are not formed un-
til after the peribranchial cavity has been completely sepa-
rated from the branchial sac. The first to appear lie far
back towards the posterior ' end, but very soon they begin to
break out in spots all over the sides of the branchial sac.

The tendency to arise in vertical rov/s becones ap-
parent, when only very fev/ are present, but eacii slit is a
separate and independent formation. I have never observed
the origin of one slit from another, such as occurs in the
larvae of Ascidians.

Pig. 5, represents a stage when about eight slits
have been formed on each side; as the anterior pouches of
the peribranchial sacs grow further and further fon.vard, new
rov/s of slits are laid down along their free margins.

The first indication of a gil]-slit is a small cir-
cular, thickened area of the branchial wall, which at this
spot becomes slightly evaginated until it touches the visce-
ral wall of the peribranchial cavity^ ^ig. 22, a. The
cells of the latter at this point become thickened somewhat,
and now a fusion takes place between the two v;alls; this is
seen in the lower portion of Pig. 22, b. The opening, which
breaks through the centre of the fused patch of cells, is
drawn out later in the long axis of the bud into a narrow
slit, which is provided v/ith cilia in the usual way. The
upper part of Pig. 22, b^, shows a slit just after the opening
has been formed.

The Branchial and Cloacal Orifices . The branchial
orifice arises at a tolerably late stage, and is first indi-

-30-

cated by a great increase in thickness of the ectoderm at a
point opposite the extreme anterior end of the branchial sac.
This thickened area becomes invaginated until the bottom of
the pit touches the endodermal wall, and a complete fiision of
the two soon takes place: Pigs. 23, a and h, and Fig. 5. The
cells in the centre break down, and the cavity of the pharynx
is put into conmunication with the outside. As is shown in
Pigs. 23, a_ and h, many mesodermal cells attach themselves
to the inner surface of the ectodermal depression, become
greatly elongated, and are eventually transformed into muscle
fibres .

In Botryllus, according to Pizon (22), it is the
branchial wall which thickens and evaginates to fuse with the
ectoderm, while the latter plays but a small part in the pro-
duction of the orifice. The irocess, as it occurs in P e r o -
phora virldis, is quite similar to that described by Kov/alev/-
sky (13) for Phallusia, and by Van Beneden and Julin (33) for
Clavelina .

The cloaca 1 orifice is formed in exactly the same
manner by the union of an ectodermal invagination v;ith the
wall of the cloaca at the anterior end of the latter, cl .o,
Fig. 5, and Pig. 20. It will not be necessary to speak here
of the various appendages and ciliated grov/ths of the pharynx
which arise later, namely, the tentacles, papillae, languets,
dorsal lamina, and peripharyngeal bands, as these are merely
differentiations of the pharyngeal epithelium and have been
sufficiently described by numerous authors.

THE DIGEST I VE TRACT.

Some time before the displacement of the inner ves-
icle is completed, and when the folds which wil] cut off the
peribranchial cavity are not very deep, the wall of the inner
vesicle high up on the left at the extreme posterior end, be-
comes much thickened, and soon evaginates to produce a little
blind pouch, the rudiment of the digestive tract. Fig. 24, d_.
tr. This lateral diverticulum grows out as a tube, which at
once bends sharply downwards and for.vards, v/hile, as the
shifting of the vesicle continues, its opening into the lat-
ter is carried up nearer and nearer the mid-dorsal line,
where it will ultimately come to lie. The tube soon turns
abruptly on itself to form a close U, and nov; grov/ing up-
wards along the outer wall of the left peribranchial sac un-
til it reaches the dorsal surface, ^i^t- finally bends directly
forward and stops short at the posterior v/all of the cloacal
cavity. At this point the distal extremity of thetube fuses
with the cloacal wall, an opening breaks through, and the
anus is established. The differentiation into oesophagus,
stomach and intestine takes place very early, and is apparent
at a stage considerably younger than that shown in Fig. 4.
The course and development of tne tube are sufficiently il-
lustrated by Pigs. 4 and 5.

As the bud grov;s and increases in length, the di-
gestive tract enlarges enormously, the U becomes opened more
and more, and the intestine describes a v;ide curve which lies

• 32-

well forward against the outer wall of tne left peribranchial
sac, Fig. 5. 'with tne anterior extension of the digestive
tract and the posterior prolongation of tne left peribran-
chial sac, the whole tract, which lies entirely on the left
side of the bud, comes eventually to be closely enveloped by
the outer wall of tne peribranchial cavity.

The "pyloric gland" or "organe refringent " of
Giard arises as a tubular diverticulum from the lower ante-
rior face of tne enlarged portion of the digestive tract
which will become the stomach. Before reaching the intes-
tine the tube bifurcates, and each branch in its turn gives
off two others, which also divide, the whole system of dich-
otomously branching tubules finally forming a lace-work sur-
rounding the whole intestine.

The development of this problematical organ is al-
ready well advanced at the stigo shown in Pig. 5, o .r . This
figure, together with Pig. 25, which shows a portion of the
stomach wall, leaves no doubt that the tube is directly deri-
ved from the digestive tract. This origin was maintained by
Delia "(''alle (3), but denied by Roule (25), who stated that
the "organe refring'nt" is not a part of the digestive
tract, but communicates v;ith the heart, and therefore belongs
to the vascular system, an opinion already held by Kuppfer.
(16)

Delia Valle's vievf is also supported by Pizon (22),
whose description of the development of the organ in Botryl-
lus , agrees minutely v;ith ray observations on Perophora.

■53-

The terminal branches of tne system of tubules
which ramify over the surface of the intestine, end in little
enlargements or ampullae, the -.vails of v/hich are very thin,
and lie closely pressed against the intestinal' wall . The
cells of the duct are cylindricil, and gradually pass over
into the flat cells of the ampullae. I have failed to find
any cilia on the latter, as Chandelon (2) has described in
Perophora. Fig. 26, shoves a cros:---3ection of the intestine
surrounded by the thin-v;alled tubules and ampullae, tne flat
cells of which contain very deeply stained nuclei. On one
side of the figure one of the ducts is cut longitudinally,
just where it forks near the surface of the intestine.

Different viev/s have been held concerning the func-
tion of the "organe refringont". Krohn /S^ , Kuppfer (16)
and Giard (G) have regarded it as a renal organ, but as the
ampullae always contain a clear, unstainable fluid, and never
concretions or epithelial debris, this view has been discard-
ed. A second hypothesis, that it is a digestive gland v/hich
gives its secretion to the intestine, has been held by Chan-
delon (2) and Delia Valle (3), the latter attributing to the
organ an hepa to-pancreatic function. Pizon (22), however,
believes that the flat cells of the ampullae possess no glan-
dular characters, and cannot be reconciled v;itn a secretory
function, but he is inclined to regard the organ as a chy-
lif erous apparatus . He says (I.e., page 95), "Je suis
plutot porte a croire que 1 '^pi theli^jm des ampoules ne se-
crete rien, et qu'il se charge simplement d'absorber les pro-

■34.

duits de la digestion qui sont assirnilables et qui n'ont pas
ete pris par les parois de I'intestin. Ces produits quit-
teraient ensuite la cellule pour aller se melanger au sang,
dont les corpuscules sont precis^ment extrernernent norabreux
autour des anpoules ter.-ninales . " Although Pizon's hypoth-
esis would seem the most probable one, as the histological
structure of the organ is not such as to suggest a glandular
function, still the role played by the "organe r^fringent"'
must remain uncertain, until the nature of the liquid con-
tained in the tubules is determined.

THE PERICARDUTI AND HEART.

Concerning the origin of the common rudiment of the
pericardium and heart investigators have given wiaely diver-
gent accounts, some deriving it from endoderm, others from
mesoderm. Although it is very certain that this structure
arises diferently in different Ascidians, still in the buds
of one and the same form statements of authors are at vari-
ance. Seeliger (29) describes the pericardium as arising in
the buds of Glavelina from an enormously large evagination
of the ventral portion of the branchial sac, which later be-
comes separated as an independent vesicle. He did not, how-
ever, distinguish the epicardial sacs, and mistook a part of
the latter for the pericardium. Van Beneden and Julin (33)
shov,red conclusively that the diverticulixm of the branchial
sac observed by Seeliger and called by him the pericardium.

-35-

is merely a part of the stolonic partition-wall and is not
concerned in the formation of the heart. According to the
Belgian authors, who described in detail the development of
the pericardi;ini in the buds of Clavelina, the pericardium and
epicardium at first form a common cavity with the inner ves-
icle. A separation takes place later in such a way that the
epicardiiim remains in communication '.vith the inner vesicle,
while the pericardium becomes entirely cut off from the lat-
ter, but retains its connection with the stolonic partition.
Van Beneden and Julin maintain that the union of the epicar-
dium with the branchial sac is never lost in the bud-develop-
ment of Clavelina, and therefore do not agree with Seeliger's
statement that the diverticulum, which he observed and er-
roneously regarded as the pericardium, beco?'ies separated from
the branchial sac. Seeliger's description of the early con-
striction of the inner vesicle from the stolonic partition is
not confirmed by the Belgians, who showed that the pericar-
diiim, originally a part of the inner vesicle preserves its
connection with the partition wall, as explained above.

A somewhat similar origin of the pericardium to-
gether v/ith the epicardium from the inner vesicle is stated
by Pizon (22) to occur in the Polyclinidae, for example in
Circinalium and Amarouoium.

Our knowledge of the derivation of this structure
in the buds of Botryllus is very much less certain. Pizon
(22) declares that the pericardium arises as a little diver-
ticulum from the lov/er wall of the inner vesicle, which be-

comes completely constricted off as an elongated tube. His
conclusion as to the endodermal origin of the peribranchial
rudiment cannot, however, be unhesitatingly accepted, since
his figures do not satisfactorily establish the correctness
of his description, while the supposition that he has not
followed the development with sufficient care, is very strong,
Salensky (27: page 527) calls attention to the fact that the
little circle of epithelial cells which Pizon marks with the
letters Per in Fig. 7, Pi.T., "wohl auch einen Ouerschnitt
der unteren Wand des Kleimendarmes darstellen kann", and that

naf a.ta.72 t>/~o t-e n /Aa.fi'/'cs

it is the same structure as the pericardii™ figured in later
stages .

The first appearance of the pericardial rudiment
observed by Hjort (8) in Botryllus, was a small clump of
cells lying against the ventral wall of the inner vesicle in
the posterior part of the bud to the right of the middle
line. As to the derivation of these cells, Hjort was unable
to say whether they were mesodermal cells or cells which had
wandered out from the endoderm, but he distinctly states that
an evagination of the inner vesicle does not occur at this
point.

In the buds of Dlstaplia Salensky (27) observed a
similar collection of cells lying against the lower wall of
the branchial sac and surrounded by mesodermal cells. He
maintains that there is no ground for attributing an endo-
dermal origin to the rudiment, which is from the beginning
sharply marked off from the v/all of the branchial sac, and

• 57-

he therefore concludes that the pericardium is derived from
the mesoderm. The result to which my observations on the
bud-developnent of Perophcra viridis have led me, in regard to
the origin of t.ie peribranchial rudiment, is in accord with
that of Salensky.

At about the stage represented in Fig. 9, a very
loose patch of cells is found applied to the outer surface of
the inner vesicle higi: up on the right side in the posterior
end of the bud. Before this time many isolated cells are
seen adhering to the wall of the vesicle at numerous points,
Figs. 7 and 6, but when the difference in thickness between
the right side and the r.- st of the vesicle is just becoming
apparent, a marked tendency in the scattered cells to acciimu-
late in one spot is noticed. At first there is but a single
layer of cells joined loosely toget/ier end to end, and forming
a somewhat elongated patch; this is the rudiment of the peri-
cardium., v^nich is the first organ to make its appearance. In
Fig. 27, u, v/hich is drawn from a frontal section, an extreme-
ly early stage is sho'.vn. That the rudiment is formed by the
coming together of free mesodermal cells , I believe there is
no reason for doubting. At the stage represented in this
figure, the similarity between many of the cells scattered
freely about in the space between the ectoderm and endoderm,
and those which form this eel] -mass, is perfectly apparent.
There is certainly not the slightest evidence that the wall
of the inner vesicle evaginates, or its cells proliferate at
this point; the line of demarcation between the two struc-

• 38-

tures is distinct throughout and shov/s no interruption in its
continuity.

The rudiment does not long remain of one layer, but
by the addition of other cells and by active cell-division it
soon becomes thicker and more compact. Fig. 27, _b. The cell-
boundaries are gradually lost, and the solid mass \s now
firmly attached to the wall of the vesicle. Pig. 27, _c. The
rudiment, which has nov/ an elongated form, does not lie hori-
zontal, but posteriorly is at a higher level than at its an-
terior 3nd .

When the shifting of the inner vesicle begins, the
clump of cells is borne pasi.ively dov/n tov/ards the ventral
side, but long before it has reached its definitive position,
a cavity has appeared in its centre, around v/hich the cells
become arranged in an epitheliiMi to form an elongated closed
sac. Pigs. 27, _d and e_.

The position of the rudiment at various stages du-
ring its descent has already been observed while considering
the displacement of the inner vesicle from Pigs. 9, 10, 11,
17 and 21.

About the time that this change in position is ac-
complished, the pericardial sac loses its attachment to the
branchial wall, and grows considerably larger and wider. The
cells composing the sac become very much flattened and atten-
uated, except in the dorsal wal] , v/hich is soon folded in
longitudinally to form the heart in the usual way. Fig. 28.
The pericardium, in its definitive position, is placed under

the posterior floor of the pharynx just to the right of the
median line. It does not lie horizontal, i.e. parallel with
the surface of the stolon, but the posterior is higher than
the anterior end. This inclination is seen in Figs. 4 and
5, in v/hich the pericardii;ra is indicated at pc . In the
latter figure, which shows about its final position, the peri-
cardium is seen to extend from a point at a level nearly as
high as the upper end of the stomach straight down to the
stalk which connects the bud with the stolon.

THE DORSAL TUBE AND GANGLION.

Of all the organs of the Ascidian bud, that which
has given rise to the greatest amount of discussion is the
nervous system. Its origin and development have been mat-
ters of much dispute, and so wide is the difference of opin-
ion concerning points of fundamental importance, that there
is little hope at present of harmonising the conflicting
statements of the various authors.

A close relation between the dorsal tube and gan-
glion has been affirmed by many v/ho hold to a common origin
of the two, but is strenuously denied by others who assert
that the dorsal tube arises independently, and has nothing
whatever to do with the nervous system. Different authors
have ascribed to these striictures an ectodermal, a mesodermal
and an endodermal origin, and have thereby exhausted the en-
tire series of possibilities.

•40-

Kowalev/sky (12), for the buds of Perophora, was the
first to describe an endoder;r,al origin of thp nervous-system.

According to him, the dorsal v/all of the branchial sac
evaginates to form a tube, v/hich retains its connection v;ith
the branchial cavity and v/hich he calls the "llervenrohr . "
In his later work on the budding of Ascidians (13) he des-
cribes the rudiment of the nervous-system in Amaroucium and
Dideninum as "ein sehr langes, an vordern Ende zeiralich brei-
tes Rohr, dessen Lumen mit der Hohle des Kiemanseokes zu com-
municiren scheint" (I.e., page 455). He did not follov/ the
development of this tube, which he held to be derived from
the endodermal vesicle, and was ignorant of its relation to
the hyphophysis and nervous-system of the adult animal. It
is probable, however, that he sav/ the ganglion in Amaroucium
at least, but failed to recognize it, for he says (I.e., page
465), "Bemerkenswerth ist noch, dass iiber dem Nervenrohr sich
eine Anhaufung von sehr blassen Zellen befindet, welche bei
'•."•eiterer Entwicklung zu verschwinden scheinen." Amaroucium
is one of those Ascidians, in v/hich the ganglion lies above
the hypophyseal tube.

Ganin (5), v;ho studied the bud-development in Pi-
demnum and Botryllus, derived the nervous-system from a ves-
icle which he described as being cut off from the inner ves-
icle of the bud, and converted into a long cylindrical tube
lying over the dorsal wall of the branchial sac. The gan-
glion, according to Ganin, becomes differentiated from a part
of this tube, the remainder of v/hich forms a ciliated organ

-41-

communicating with the branchial cavity. 'His description is
very obscure, however, and the only points to be noticed are,
that the dorsal tube, accordinr: to this author, is derived
from the endodermal vesicle, and that it gives rise to the
ganglion.

Giard (6) and Delia 7a lie (3 and 4) v/ho studied the
bud-development in different species of Ascidians, contribu-
ted nothing of value concerning the nervous-system, but both
ascribe a common origin to the dorsal tube and ganglion.

The viev/s of Seeliger (29) are very different from
the foregoing. According to hi:., the dorsal tube and gan-
glion in the buds of Clave lina arise from a common rudiment,
which is derived from mesodermal cells. This belief was not
based on direct observation, since he did not examine suffi-
ciently young stages, but was arrived at through theoretical
considerations. The great similarity between the individual
cells of the nerve-rudiment and the free mesodermal cells in
the body-cavity of the bud, Seeliger holds in good evidence
for the mesodermal origin of this structure. He furthermore
points out that the cells composing the ganglion of the lar-
va, would be carried off in the blood after the disintegra-
tion of that organ, ana give rise in the bud to some of the
mesodermal cells. The latter would therefore be "directe
Abkcmmlinge eines friihere gangliosen Organs", and it would be
but natural for them to resume the function which they had
once possessed. Van Beneden and Julin {33) in their work
on the development of the buds of Clavelina, state, that the

• 42-

nervous-system is derived from the ectoderm, and first ap-
pears as a cord of cells lying close against the ectodermal
wall. Their description, however, is very incomplete and
unsatisfactory.

Our more recent knov^rledge of the subject is due to
the researches of Pizon (22), Oka (20), and Hjort (8) on Bo-
tryllus, of Salensky (27) on Distaplia, and of Gaullery (1)
on Glosophorim and Diplosona. The first tnree authors, al-
though they are in agreement concerning the origin of the
dorsal tube, differ widely in respect to the derivation of
the ganglion. According to all tnree, the dorsal tube in
the Botryllus bud arises as an anteriorly directed evagina-
tion of the dorsal wall of the branchial sac, ending blindly
in front, but freely opening into the branchial cavity at its
posterior extremity. This tube grov/s forward, and its ante-
rior end fuses with the wall of the branchial sac, whose
cavity is then put into communication with the lamen of the
tube, while the posterior connection becomes obliterated.
The definitive opening of the hypophyseal tube is therefore
secondary. So far v/e find these authors agree, but it is
quite otherwise when we come to consider the origin of tne
ganglion.

Pizon maintains that the ganglion of the bud is de-
rived directly from a fine nerve-string, which grov/s out from
the ganglion of the parent-bud, or in the first from tnat of
the larva, into the young bud. His view is not based on ac-

43-

tual observation, and his arguments, which are far from sat-
isfactory, fail to convince. He avers that a constriction
of the ganglion from the wall of tne dorsal tube does not
take place, but, on the contrary, the figures of Kjort (8:
Botryllus) prove fairly conclusively that such a constriction
does actually occur. Kjort 's contention that the ganglion
is formed from the thickened ventral wall of tne hypophyseal
tube, is based on a study of an unbroken series of stages and
is clearly borne out by his figures. In a short note""" on
the budding of Botryllus, v/hich was published recently, I
added additional evidence in support of Hjort's view, and re-
produced a drav/ing v/hich showed beyond a doubt that the
thickened ventral v/all of the dorsal tube is pinched off to
form the ganglion. The account given by Oka is entirely
different. According to this observer, cells wander out
from the ectoderm, fasten themselves to the ventral wall of
the dorsal tube, and there form tne ganglion. These wander-
ing ectodermal cells were also observed by Pizon, who descri-
bed them as giving rise to a portion of the genital gland, to
muscular fibres, and to certain cells of the blood. The
principal difference, then, bet'.veen the three authors is,
that whereas Pizon and Oka hold to an independent origin of
hypophysis and ganglion, Hjort maintains that there is a com-
mon rudiment for the tv/o structures.

The results of Salensky (27) on the bud-development

Johns Hopkins University Circulars, No. 119, June, 1895.
-44-

of Distaplia do not stand in the slightest agreement with
any of those obtained for Bo_tryllus. The nervous-syste.'n of
the Distaplia-bud is of ectodermal origin, according to him.
Cells sink down at a very early period from the ectodermal
wall, and form a solid mass, which later acquires a cavity,
increases in length, and produces a tube. The latter be-
comes differentiated into three parts; the anterior gives
rise to the hypophysis, the middle to the ganglion, and the
posterior portion to the visceral nerve. The hypophysis and
ganglion have, therefore, a common origin. This mode of
formation, however, is only true of the primordial bud; for
all the other buds, which are produced from it by fission, de-
rive their nervous-system by division directly from that of
the parent along with the rtst of their organs.

Tt might be mentioned that in the buds of Pyrosoma,
Salensky (26) has described a similar ectodermal origin of
the nervous-system, although Seoliger (30) in the same form
aerives the common rudiment of ganglion and hypophysis from
mesodermal cells.

Salensky's results on Distaplia are directly con-
tradicted by Hjort and Frl . Bonnevie (10). The latter find
no trace of the nervous-system in the early stage at which
Salensky describes its first appearance, but maintain, on the
contrary, that a forwardly directed diverticulum is later
formed from the dorsal wall of tne inner vesicle, just as in
Botryllus, and that the ganglion is differentiated from the
wall of the dorsal tube.

-45-

Pizon (22), in his v/ork on Botryllus , states that
he has made observatio s on the develop'nent of the dorsal
tube in the buds of a number of other Ascidians. In Perq-
phoraand Clave lina he observed tne tube over the dorsal v^all
of the branchial sac, but did not obtain stages which were
young enough to enable him to determine its origin. He con-
cludes, however, on the insufficient evidence of Kovmlev;sky 's
observations on Perophora, that the dorsal tube arises as a
diverticuluiTi of the endoderrnal wall. Since in Glavelina the
later stages in the development of the dorsal tube are simi- .
lar to those of Perophora, he holds that in this Ascidian al-
so the origin is the sane. In two of tne Polyclinidae, viz:
Amaroucium proliferurn and Circinalium, in in Didemnurn niveun;
and in Astellium sponglf orrne, he has observed the dorsal tube
arising as an endodermal diverticulaTi, v/hich acquires a sec-
ondary opening into the branchial sac at its anterior extrem-
ity, just as in Botryllus .

In none of tnese forms did he determine the origin
of the ganglion, but he comes to the unwarranted conclusion
that this structure is derived, independently of the dorsal
tube, in the sane way as he has described for the Botrylli-
dae .

Hjort (9) has recently studied the development of
the neuro-hypophyseal system in the buds of glossophormi sa-
bulosum, one of the Polyclinidae , and Caullery ( 1) in Glos-
sophorum l\iteurn, Gircinali;m concrescens and Diplosoma gela-
tinosum, and although both of these authors find that the

-46-

dorsal tube arises in the manner described by Pizon, that is,
as an anteriorly directed endoder.-aal diverticulum, they give
a different account of the origin of tne ganglion. . In all
the species studied, tne ganglion is formed as a differen-
tiation of tne dorsal v/all of the hypophyseal tube, and has
therefore a comnon rudiment v/ith the latter. Their results
are in agreement v/ith vj-hat Hjort has found in Botryllus, ex-
cept that in all of the above-mentioned Ascidians the hypo-
physis lies belov; the ganglion, v/hereas in Botryllus it is
above .

Finally, Ritter (24) who has recently described the
bud-development of Goodseria, a genus in which budding had
not been observed before, finds a complete agreement, concern-
ing the origin of the neuro-hypophyseal system, with Hjort 's
work on Botryllus .

In the same paper Ritter gives a preliminary ac-
count of some observations on the development of the buds of
Perophora anne^ctens (Ritter) and P. Iiis.tejM^- ^^^ both spe-
cies he derives the common rudiment of the hypophysis and
ganglion from cells which wander out from the dorsal wall of
the inner vesicle.

After this short review of the state of our know-
ledge regarding this much confused subject , I shall now give
an account of my ov/n observations on the development of the
dorsal tube and ganglion in the buds of Perophora viridis_.

My results, which are based on a study of an unin-
terrupted series of stages, have led me to believe that the

•47-

conclusion :';hich Seeliger drev/, from purely theoretical con-
siderations, concerning tne origin of tnese structures in
Clavelina is also true of Perophora viridis. I shall try
to show tnat tnere is every reason to believe that tne dorsal
tube and ganglion are derived from .'7iesoder:nal cells .

The dorsal tube is formed long before the ganglion ,
and the rudiment from which it v/ill arise is first indicated
just after the collection of cells v/hich is to produce the
pericardiam makes its appearance. 'Vhen the difference in
thickness between the right side and the rest of the vesicle
is becoming apparent, hence at a time --.vhen tne rotation is
about beginning, in the anterior portion of the bud, a little
to the left of the median dorsal line, there is seen an ir-
regular elongated patch of cells very loosely grouped togeth-
er and lying on the outer surface of the inner vesicle, Pig.
28, d^.t. The free mesodermal cells in the space between the
ectoderm and endoderm are especially numerous in this region,
and are closely associated with the collection of cells ad-
hering to the vesicle. Although I have examined my sections
with the greatest care under a ■'"/12 oil-i.'nmersion lens, from
the very first appearance of the rudiment, I have failed to
find any indication of cell-migration from the endodermal
wall, and therefore cannot confirm Hitter's statement (I.e.
page 368) that an "indistinguishable transition from the
cells of the 'endoderm' to those of the neuro-hypophyseal
anlage is to be traced", and also that cells can be found
"in the act of migrating from the "endoderm" into the anlage."

-48-

The line of separation between the rudiment and the
wall of tne vesicle is seen to be perfectly distinct and
clearly marked, and there is no evidence whatever of prolif-
eration of endodermal cells at any point; Fig. 29, a. Du-
ring the early stages of development the cells which are to
form the dorsal tube, and many of the mesodermal cells, are
absolutely identical in appearance, and exui.At the same
amoeboid character. So gradual is the transition from the
free mesodermal cells to the cells of the rudiment, that it
is at first impossible to say where the former end, and the
latter begin, Pig. 29, b.

I believe, therefore, that there is no evidence
whatevejr for holding that the dorsal t_ube does not aris_e frojn
free mesodermal cells of t^he blood.

By further additions from outside and by active
cell-multiplication -.vithin the mass, the rudiment gradually
increases in size; its cells become more closely packed to-
gether, and soon form an elongated solid cord lying close
against the dorsal wall of the vesicle in the anterior end
of the bud. Fig. 29, c^. Fig. 29, a_, _b and _c illustrate the
development up to this point; a is drawn from the same sec-
tion as Fig. 28, h and _g from the series to which Figs. 10
and 11 respectively belong. It has been stated above that
the anterior portion of the inner vesicle is only slightly
involved in the displacement already described, but that it

is to a certain extent, is proved by the fact that the rudi-

first appears
ment of the dorsal tube not exactly in the mid-dorsal line,

A

■49-

but a little to the left of this, Pig. 23. 3y the time the
shifting of the vesicle has proceeded soraev/hat further than
is shown in Fig. 11, the string of cells, which is now solid,
has been carried up to the median plane.

Very shortly after it has reached its definitive
position, a lumen appears in the centre of the rudiment
throughout its entire length, and around this the cells be-
come arranged into a one-layered epitheliiim. Pig. 27, d_ and

By following the course of development up to this
point, it is seen hov/ an epithelial tube does actually arise
from free mesodermal cells, a thing which Hjort has charac-
terized as most improbable. In criticising Seeliger's view
of the origin of the neuro-hypophyseal system in Clave lina,
this author says, (8: page 602) "die Wahrscheinlichheit da-
fur, dass ein Ganglion und ein epitheliales Rohr sich aus
zusammengehauf ten Mesodermzellen bilden sollte, scheint mir
so gering zu sein, wie fur die Auffassung Herdman's dass die
Innere Blase der Knospenanlage einen solchen Ursprung habe . "

About the time that the peribranchial cavity is
completely cut off from the inner vesicle, the anterior ex-
tremity of the dorsal tube fuses with the dorsal wall of the
branchial sac, an opening breaks through, and the lumen of
the tube is put into communication with the branchial cavity.
The posterior end of the tube abuts against the anterior
wall of the cloaca, but neve-.r opens into the latter, in con-
trast with the condition found in Botryllus and many other

-50-

Ascidians .

Fig. 50, represents a median sagittal section of a
bud before the complete separation of the peribranchial cav-
ity, and therefore before the dorsal tube has acquired an
opening into the branchial sac. The section passes through
the entire length of the tube, d . t_, v/hich is seen to be clo-
sed at both ends and made up of an epithelium of one layer.

In my preliminary work on the budding of P.erophora,
already referred to, I made the statement that, "the ganglion
is formed by a thickening of the d_orsal wall of the tube,
which eventually becomes constricted off in the manner des-
cribed by Kjort for Botryllus, although in the latter it is
the _ventral v/all of the tube which gives rise to the gan-
glion." More careful study of very young stages, however,
has convinced me that the above is not an accurate descrip-
tion of the formation of the ganglion.

After the communication between the dorsal tube and
branchial sac has been established, a few cells, identical
in appearance with mesodermal cells, are found adhering to
the dorsal surface of the tube throughout the greater part of
its length; this elongated, loose patch of cells constitutes
the rudiment of the prur-licn, £l. Fig. 31, a_.

It is a difficult question to decide whether these
cells are entirely mesodermal, as their appearance indicates,
or whether they are derived from proliferation oi' tne wall of
the tube, fot- in many places the outline of the latter is
broken, and there is no sharp demarcation between the cells

-51-

of the rudinent and those of the tube, as seen in Pig. 51, a.

]"[any sections, however, such as the one shov/n in
Fig. 31, _b, v/hich represents a slightly older stage, leave
little room for doubt that nuclei do wander out into the ru-
diment. But, on the other hand, I think that this figure
shows equally well that mesodermal cells are added to the
mass from the outside.

I have, therefore, come to the conclusion that the
ganglion has a double origin, and that both the wall of the
tube and free mesodermal cells co-operate in forming it.
It is to be remembered, however-, that ultimately it is entire
ly a mesodermal structure, as we have seen hov/ the dorsal
tube is constructed out of cells of the blood.

The ganglionic- rudiment is at first a very irreg-
ular heap of cells, and is clearly associated at the periphe-
ry with surrounding mesoderm.al cells. The cell-boundaries
are completely lost very early, and the mass rapidly increases
in size by multiplication of nuclei within, by further ac-
quisition of cells from without, and by continued migration
of nuclei from the wall of the tube. Pig. 31, c. The nuclei
now arrange themselves in a couple of layers around a central
core, in which fine fibrils are laid dov/n, and the ganglion
becomes completely marked off from tne v/all of the tube; the
definitive cti'ucture is nov/ attained.

'"ig. 31, d and _e, illustrates the latter course of
development .

?;Iy observations, therefore, have led me to believe

that the hypophyseal tube and the ganglion are formed only
in part from a common rudiment, and in this respect to take
a middle ground between Kjort, Salensky, and Caullery, on the
one hand, who have described a common origin for these struc-
tures in the Ascidians studied by them, and Oka and Pizon on
the other, who maintain that they arise independently. In
deriving the hypophysis and ganglion, hov/ever, from tne mes-
oderm, I differ widely from all previous observers with the
exception of Seeliger. Concerning the origin of the dorsal
tube in the buds of Perophora my results are totally opposed
to the conclusion of Pizon, which as stated above, is not
based on sufficient evidence, namely, that "le tube dorsal
des Perophores a la meme evolution que le tube dorsal des Bo-
tryllides", (I.e., page 130).

A study of the younger stages would have convinced
him of his error.

Perophora viridis, at all events, presents an ex-
ception to the general rule laid down by Pizon, that "Chez
toutes ces families d'Ascidies composees (Clavelinidae, Pero-
phoridae, Botryllidae, Polyclinidae, Distomidae, Didemnidae,
et Diplosomidae ) I'organe vibratile debute par un tube aveu-
gle, forme par un diverticule de la vesicule endodermique
primitive." (I.e., page 131.)

THE SEXUAL ORGANS.

:.!y observations on the development of the sexual

organs have not been carried beyond quite an early stage, but
so far as they go, they clearly agree with the description

given by Van Beneden and Julin (33) for the buds of Peropho-
ra Listeri .

Shortly after the peribranchial cavity has been
completely divided off from the branchial sac, a small col-
lection of cells appears between the two arms of the U-shaped
digestive tract and at the level of the duct of the "organe
refringent", almost at the point where the latter is connect-
ed with the stomach. The sexual organs arise from this lit-
tle sp?ierical mass of cells, which are at first but loosely
held together and identical in appearance with the mesodermal
cells of the blood. ?ig. 32, a, ./hich is precisely similar
to Van Beneden and Julin 's Pig. 5, b^, PI. XVI, shows the in-
timate relation between the cells of the blood and those of
the rudiment, so that there can be no doubt that the latter
are of mesodermal origin. The connection, already described
by the Belgian authors, of some of the peripheral cells of
the mass with surrounding mesodermal cells by protoplasmic
processes, is distinctly seen in the figure. A small ir;reg-
ular cavity is also shown in the centre of the clump of cells'-

Some of the free mesodermal cells lying above the rudiment,
g . c . Pig. 32, a, are seen to be spindle-shaped; these soon
become joined, end to end, to form a solid cord united at
one end to the spherical mass of cells, and taking a course
parallel to that of the intestine, g.c . 32, b. This figure
represents a later stage, in which the cavity is considerably

-54-

enlarged and the genital cord, g^.c, is present as a solid
single rov; of cells, (Compare Fig. 2, Pl.XI-I, of /an 13ene(3en
and Julin. )

A furrow, which appears opposite to tne attachment
of the cord, novr divides the hollow sphere into two lobes,
the cavities of which are not completely separated, but re-
main in communication. In Fig. 32, c^, the division of the
originally simple sphere is seen, but the section is not in
the proper plane to show the connection between the two cavi-
ties .

One of these lobes gives rise to the testis, the .
other to the ovary, according to Van Beneden and Julin, who
have described in detail how, from the primitive, simple
sphere and the single cord of cells, testis, ovary, vas def-
erens and oviduct are all differentiated.

Ily observations would therefore seem to support the
view of the Belgian authors that the male and female sexual
organs do not arise from separate vesicles, as described by
Kov/alewsky (12) in the buds of Perophqra, but are formed from
one and tne same rudiment.

SUr.CvIARY OF RESULTS.

1. The ruciiment of the Perophora bud, like that
of all other Ascidians, consists of two vesicles, an outer
and an inner one. The former is derived from the ectoderm
of tne stolon, tne latter from the thickened evaginated wall

■55-

of tne stolonic partition.

2. At an early stage the right side of the inner
vesicle is found to be much thicker t/ian the remaining por-
tion, and by a peculiar process of rotatory growth or dis-
placement of the vpsicle, is carried dovm to the ventral side
of the bud, where it forms the floor of the pharynx. This
process seems to be due to tne growth and flattening out of
the cells composing the whole wall of the vesicle except in
the thickened region. I am at a loss to explain why the dis-
placement of the vesicle should occur, and to discover the
phylogenetic significance of it, if it have any.

3. The peribranchial sacs arise asymaetrically .
As tne displacement proceeds, the wall of the inner vesicle
is folded in, at the point where the right side of the vesi-
cle joins the stolonic partition, to form the le_ft peilV°^?.5."
chial sac. The connection of the latter with the partition
of the stolon is retained until a much later stage. A lon-
gitudinal furrow appearing high up on the right side of the
inner vesicle, separates off tne right peribranchial sac, and
is gradually borne ventrally, as the shifting of the vesicle
continues. The constriction of the whole peribranchial cav-
ity eventually takes place in such a way as to cut off from
tne peribranchial sac a :)-shaped bag, composed of the median
dorsal connecting piece or cloaca, and the two lateral por-
tions of the cavity. Anterior and posterior extensions of.
the latter gro\7 out and surround respectively the anterior
and posterior ends of the pharynx. The posterior prolonga-

tions are possibly to be regarded as ho.'nologous \7ith the epi-
cardial sacs of some other Ascidians.

4. The endostyle appears early as a longitudinal
groove in the middle of the thickened portion of the vesicle;
from its primitive position on the ri^ht side it is moved
dov/n to tne ventral mid-line by thp displacement of the ves-
icle.

5. The digestive tract grows out laterally as a
blind tube from the posterior end of the inner vesicle high
up on the left side. During the change in position of the
vesicle, its opening into the latter is carried up into the
median plane.

The "organe refringent" arises as a tubular diver-
ticulim from the anterior face of the stomach, and produces
a dichotomously branched system of tubules, which surround
the intestine and terminate in little dilated vesicles or am-
pullae .

6. All the evidence goes to show that the peri-
cardium is formed from free meso.iernal cells. It first ap-
pears as a clump of cells adhering to the outer surface of
the inner vesicle far wp on the right side, and through the
shifting of the vesicle is brought down to the ventral side.

7. The dorsal tube and ganglion are formed only
in part from a common rudiment, and there is every reason to
believe tnat both are derived solely from tne mesoderm. The
former appears as an elongated, solid mass of cells, which
lies close against the outer surface of the inner vesicle,

a little to the left of the median dorsal line. '■:rnen the
displacement of the vesicle is completed, the rudiment lies
in the Tiedian plane; it then acquires a lumen, which is put
into communication anteriorly v/ith the cavity of the pharynx.
The ganglion has a double origin, and is formed by prolifer-
ation of the upper wall of the dorsal tube and also from free
mesodermal cells.

8. The sexual organs have a comiaon mesodermal or-
igin, both testis and ovary arising from one and the same
spherical rudiment.

C ONCLUD I NG REMARKS .

The results which I have obtained from the study
of the budding of P_erophora viridis furnish additional evi-
dence in support of the viev/ that the development of the bud
and that of the larva do not proceed along parallel lines.
The attempts which have been made to harmof||inise the facts
of budding with the germ layer theory, have benn totally fu-
tile in the cuse of Ascidians, and ariy hypofiasis "Jhi':'\ ex-
plains budding in the Ascidians as a procesr. of regeneration ,
by which the organs of the parent, or their-layers , give rise
to sim.ilar organs in the bud, must, in the light of known
fc.'Cts, be ruled out.

The rudiment of the bud in all groups of Compound
Ascidians is composed of two vesicles, one wi*:hin the other,
enclosing between them free mesodermal cells. The outer

• 58-

vesicle Is always derived from the ectoderm of the parent,
and gives rise to the ectodermal covering of the bud. The
origin of the inner vesicle, however, is not the sane in all
Ascidians. In the Botryllidae it arises, in both larva and
bud, from the perlbranchial wall, which is formed in the first
place from, the ectoderm of the embryo. This vesicle is,
therefore, ultimately of ectodermal ori^^in in Botryll us ,
whereas in al] other Ascidians it comes from an endcdermial
structure of the parent.

Although derived in the two cases from different
germ-layers, the inner ve'sicle may go to form the same organs
in the bud, but organs which are of widely different origin
in the larva; for example, the digestive tract and nervous
system. (Pljort, Botryl] us (8), Distaplia (10), Glcssophorum
(9).

The fate of the inner vesicle is consequently di-
rectly opposed to the application of the germ-layer theory in
its ordinary meaning to the Ascidian bud.

In Perophora viridis the important part played by
cells, which there is every reason to regard as mesodermal
cells, in the bud-development, only increases the disagree-
ment. Organs which in other Ascidians are formed from ec-
toderm or endoderm, are here of mesodermal origin. The ner-
vous-system and pericardium, v/hich are respectively ectodermal
and endodermal productions in the larva, develop out of free
mesodermal cells.

Since in the development of the bud and larva the

the sane end is reached by entirely different roads, and in
the foi-mer organs do not proceed from corresponding organs,
or even their germ-layers, in the latter, and moreover, since
in the bud a rudiment derived from one and the sam.e larval
germ-layer may give rise to structures of widely different
nature, one is compelled to believe witn Hjort, that "die
Knospen der zusanmengesetzte Ascidien ein Entv/icklungsprocess
ist, in v/elchem sam.tliche Organe durch 'Neubildung' aus einer

sehr primitive Anlage entstehen." "Die Knospe muss

ihre eigenen Gesetze haben und muss, da sie aus einer wesent-
lich anderen Anlage hervorgeht als die Larve, auf andere
Weise gebildet werden. Ebenso wie dass Ei, m.uss das :iate-
rial, die Anlage, welche den Ausgangspunkt fur die Entwick-
lung bildet, als ganz undifferenzirt gedacht werden und muss
alles enthalten, was zur Bildung eines ganzen Individuimis no-
tig ist, ebenso wie die Blastula des Eies," (9:, page 225).

The behaviour of the 'm.esooerm' in the bud-devel-
opment of Perophora viridis is of much interest, ov/ing to the
number and variety of organs in whose formation nesodermal
cells are concerned. As we have seen these cells give rise
to the pericardium, the dorsal tube, the ganglion, and the
sexual organs; they also produce the musculature, and, as
Kowalewsky (14) and Seeliger (32) have shown in other Asci-
dians, in Perophora also they doubtless become the cells of
the cellulose test.

I have spoken of the cells which go to form the
pericardium, nervous-system, etc., as 'mesoderm.al • , because

-60-

they are identical in appearance v/ith the amoeboid cells
which are scattered about abundantly in the body-cavities of
tne buds and the sinuses of the stolon. As I have emphasis-
ed repeatedly in describing the formation of tne various or-
gans in question, there is not the slightest discoverable ev-
idence that cells are given off from the wall of the inner
vesicle at the points where such organs arise, or at any oth-
er place, for that matter, and this fact, together with the
similarity betv/een the free mesodermal cells and the cells
which go to make up these rudiments, is strong proof that the
latter are genuine mesodermal cells.

The origin of these cells is a question which is

practically undeterminable, and direct proof that they are

of tne mesoderm
all descendants of the larva, is of course impossible, al-

though the assumption that they are, seems to me to be a jus-
tifiable one. The statement that they do not arise from
cells which wander out from the endoderm or ectoderm, is ba-
sed, it is true, on negative evidence, since after the most
diligent search it cannot be discovered that any are derived
in this manner.

It is, perhaps, going a little far to absolutely
deny such an origin, for it would be impossible from the na-
ture of the case to say that at no time does the endoderm or
ectoderm give off cells to the blood, even although it cannot
be shown that such is the fact.

The positive evidence furnished by their mesodermal
appearance, however, together with the negative evider. ce de-

rived fron the entire absence of any indication of another
origin, almost amounts to a certainty that these cells are
truly mesodermal.

If then this assumption be admitted, a discussion
of the nature of these cells and the part played by them
in the developing bud is not out of place.

In the case of a fixed organ, like the ganglion or
pericardii.m, which has a definite and determined position,
the cells destined to form it, must by virtue of their motile
power come together at the right time and place.

Weismann (35; pages 161-52) in discussing the pro-
cess of gemmation in Glavelina, as described by Seeliger (29),
supposes that t.-ie nescdermal cells "contain very different
kinds of idioplasm: ox\p , for instance, might contain 'muscle
determinants', and another, 'nerve-determinants', and a third
•blood-corpuscle-determinants'." He further adds, that
"until we knov/ more of the actual facts concerned, we can on-'
ly -- ho\7ever unsatisfactory such an assumption may be -- at-
tribute to the cells a tendency to become attached at defi-
nite points according to the manner in which they have pre-
viously been determined."

It seems to me, however, that the opposite assump-
tion, which Weismann regards as less likely, namely, "that
these cells develop into muscle, nerve, or sexual cells
according to their point of attachment," is more in accor-
dance with the facts presented by the bud-development of Pe-
rophora viridis.

We have already seen in the very young bud, when
it consists nerely of two sinple layers, and before there is
the slightest indication of the appearance of any organ, that
mesodermal cells are attached in many places to the inner
surface of the ectoderm, and outer surface of the endoderm,
but that they are nowhere more numerous at any one spot than
another, Pigs. 7 and 8. Tneir poY,rer of amoeboid movement
over any surface v/ith v/hic;T they come in contact, would ac-
count for their presence on the walls of the vesicles.

It would seen more probable that these cells are
all alike and undifferentiated, and that the nature of the
organs to which they give rise is determined, not by any pre-
arranged condition of their idioplasm, but by the particular
point to which they happen to become attached. I regard it
as a significant fact that mesodermal cells are found, not
only at the places v/here organs will arise, but also at many
other points. Those of the cells which chance to fall, as
it were, on fertile soil, will undergo further development,
and under the formative influence exerted upon them by that
portion of the wall to which they adhere, will be utilised in
building up a definite structure.

All parts of the walls cannot possess a specific
determining po'.ver, and such cells as lodge on barren ground,
are not further modified, and do not furnish material for the
formation of organs.

According to this viev/, one mesodermal cell is the
equivalent of any otner, and it is only a few that find fa-

vorable positicns, and have their latent possibilities called
forth.

Those which become attached at a point high up on
the right side of the inner vesicle in the posterior region
of the bud, v.'ill forin the pericardiim; others on the dorsal
side, at the anterior end, v/ill give rise to the dorsal tube,
and still others, v/hich lodge on the upper wall of the latter,
will help to construct the ganglion; sorrie adhere to the in-
ner surface of the ectoderm, lengthen out and become muscle-
fibres, some wander through the ectoderm, and on the external
surface are transformed into the cells of the cellulose test,
while others find a definite place in the posterior region
of the bud and develop into the sexual organs.

This viev/ is opposed to the supposition of Seeliger
spoken of above, that in Clavelina the ganglion of the bud is
formed from free cells of the blood, v/hich had earlier com-
posed the larval ganglion and been liberated on the dissolu-
tion of that organ; these cells would, therefore, have al-
ready possessed a ganglionic nature, and would merely resume
in the bud their former function.

On any such assumption, it is almost impossible to
imagine how isolated specific cells, moving freely about in
the blood, could reach their proper destination. On the re-
verse assumption, hov/ever, the presence of rnesociermal cells
at any particular point is accidental, but once there, their
potentialities are called out under the specific formative
influence of the place of attachment.

Baltimore, Id., April 18, 1696.

LIT'^RATURE CITKD.

1. Caullery, Li.

2. Chandelon, T. --

3. Delia Valle, A,

4. Delia Valle, A. --

5. Ganin, M. -■

6. aiard, A. --

7. Herdaan, Y/.A.

8. Hjort, J.

Contributions a I'etiide des Ascidies
composees. -- Thesis, Paris, 1895.
Recherches sur line annexe du t;ibe di-
gestif aes Tuniciers. -- Bulletin
Acad. Royale de Belgique, T. XXXIX,
1875.

Recherches sur I'anatomie des Ascidies
composees. --Arch. Ital. Biol. T. II,
1882.

Sur le bourgeonnement des Didernnides
et des Botryllides. -- Arch. Ital. Bi-
ol. T. TI, 1882.

ITeue Thatsachen au.s der Rntwicklungs-
gGschichte der Ascidien. -- Zeitsch.
wiss. Zool. XX Bd., 1870.
Recherches s-ar les Ascidies composees
ou Synascidies. -- Arch. Zool. Rxper.,
T. I, 1872.

Report on the Tuniccita. -- Challenger
Reports, Vol. VI, 1882.
Uber den Entwicklungscyclus der zu-
samrnengesetzten Ascidien. --- i.Iittu.
zool. station Neapel, X, 3d., 1893.

9. Hjort, J. -- Beitrag zur Keirnblatterlehre und Ent-

wicklungsinechanik der Ascidienknospung
-- Anat. Anz., X Bd . , No. 7, 1894.

10. Hjort, J. und Prl . Bonnevie. — Uber die Knospung von

Distaplia jaagnilarva. -- Anat. Anz.,
X Bd. , 'lo. 12, 1895.

11. Kovmlewsky, A. -- V»'eitere Studien u?jer die Entwicklung

der einfachen Ascidien. -- Arch. nikr.
Anat., VTT 3d., 1871.

12. Kowalewsky, A. -- Sur le bourgeonne?rient du Perophora

Listeri. -- (Trans, from the Russian,
by A. Giard) -- Rsv. sc . nat. "lont-
pellier, T. I[[, 1874.

13. Kov7alev/sky, A. -- Uber die Knospung der Ascidien. - =

Arch. mikr. Anat., X Bd . , 1874.

14. Kowalev/sky, A. -- Einige Beitrage zur Bildung des Man-

tels der Ascidien. -- 'Tem. kc . Sc . 3t.
Petersb. T. 28, 1892.

15. Krohn, A. -- Uber die Fortpf lanzungsverhaltnisse

bei den Botrylliden. -- Arch. Naturg.,
XXXV Bd., 1859. -- Uber die fruheste
Bilding der Botryllusstocke . -- Ibi-
d era .

16. Kuppfer, C. -- Zur Entwicklung der einfachen Ascidien

-- Arch. mikr. Anat., VIII Bd . , 1872.

17. Lahille, P. -- P.echerches sur les Tuniciers des cotes

de Prance. -- Tnesis, Toulouse, 1890.

-55-

18. Iietschnikoff , E. --Uber die Laven und Knospen von Botryl-

lus . — Bull. Ac. f;C. St. Petorsb.,
T. 1:3, 18o9.

19. Metschnikoff , E. --Enbryorialentwicklung der einfachen As-

cidien. -- Ibidem.

20. Oka, A. -- Uber die Kncspimg der Botrylliden.-

Zeitsch. wiss. Zool., LIVBd., 1892.

21. Patten, V/. -- Orienting small objects for sectioning^

and fixing tnem when moiinted in cells.
-- Zeitsch. wiss. Mikr., XI Bd . , 1894.

22. Pi7.on, A, -- Histoire de la blastogenese chez les

Botryllidps. -- Ann. Sc . Nat., Zool
(7), T. XIV, 1893.

23. Ritter^ W.PJ. -- Tunicata of the Pacific Coast of North

America. 1. Perophora annectens, n.sp.
-- Proc. California Ac. Sc . , Vol. IV,
1894.

24. Ritter, -".E. -- On biidding in Goodsiria and Perophora.

-- Anat. Anz., X Bd . , No. 11, 1895.

25. Roule, L. -- Recherches sur les Ascidies simples

des cStes de Provoiice (Phallusiadees ) .
Ann. 'ius. Nat. Hist. Marseille, T. II,
1884.

26. Salensky, V/. -- Beitrage zur Srnbryonalentwicklung der

Pyrosomen. -- Zool. Jahrb. Abtn. f.
Anat., IV, VKd., 1891, 1892.

27. Salensky, W. -- Morphologische Studien an Tunicaten,

-57-

II. -- :iorph. Jahrb. XX Bd . , 1893.

28. Salensky, v/. -- Beitrage zur Entwicklungsgeschichte

der Synajcidien T, IT. -- Mitth. zool.
Stat. TTeapel, XI Sd . , 1894, 1895.

29. Seeligftr, 0. -- Eiblldung und Knospung von Clavelina

lepadif orinis . -- Sitz. Ber. Akad ,
Wien, 'latn.-- "Taturwiss. Classe, LXXXV
3d. , T Abth. , 1882.

30. Seeliger, 0. -- Zur Entwicklungsgescaichte derPyroso-

men. -- Jena. Zeitsch. TTaturwiss . ,
XXI I [ Bd. , 1889.

31. Seoliger, 0. -- Uber die Kntsteh' ng des peribranchia-

len Raunen bei den Ernbryonen der Asci-
dien. -- Zeitsch. wiss. Zool., Il/I,
Bd., 1893.

32. Seeliger, 0. -- Einige Beobachtungen uber die Bildung

des ausseren Mantels der Tunicaten. -
Zeitsch. v/iss. Zool., LVI Bd . , 1893.

33. Van Beneden et Julin. -- Recherches sur la morphologie

des Tuniciers. -- Arch, de Biologie,
T. VI, 1887.

34. verrill, A.E. -- Brief Contributions from tiie Museum of

Yale College. No. XVI.
On the Distribution of Marine Animals
on the Southern Coast of ITe\7 England.
Amer. Journ. Sc . and Arts, (ser.3)
Vol. IT, p. 359, 1871.

35. v/eismann, A. -- The. Crerm-plasm. (Trans.) wew York,

1893.

36. '.Villey, A. -- Studies en the Pro tochordata, I, II.

Quart. Journ. micr. Sc, Vols. XXXIV,
XXXV, 1893.

-69-

EXPLANATIOK OF THE FIGURES.

All the drawings have been made with a camera luci'
da. The lenses used viere those of Zeiss, and are referred
to in the description of each figure.

Reference Letters .

a_. Anus.

amp . Ampulla of the "organe refringent."

bl . s. Blood-sinus of stolon.

br . Branchial sac.

br . 0^. Branchial oi'ifice.

_c. g. Cavity of rudiment of sexual organs.

c 1 . Cloaca.

c3^._^. Cloacal orifice.

£. p_c. Cavity of pericardial sac.

jc. t. Cells of test.

d . t . Dorsal tube.

a_. jtr. Digestive tract.

d.w.p£. Thickened dorsal wall of pericardiT-ra, which becomes

invaginated to form the heart.

£.br._o. Ectodermal invagination to form the brancnial orific(

— ■Sl'S-' Ectodermal invagination to form the cloacal orifice.

ect . Ectoderm,

en. Endoderm.

end. Endostyle.

-70

£._l.p_b._c. Fold to form left peribranchial cavity.

£.r.pb._c. Fold to form right peribranchial cavity.

g._c. Gienital cord.

gl . Ganglion.

g. _s. Crill-slit.

int. Intestine.

l.a.ex. Left anterior extension or pouch of peribranchial
cavi ty .

1. d. t. Lumen of dorsal tube.

l.pbr.c. Left peribranchial cavity.

l.p.ex. Left posterior extension or pouch (left epicardial
sac?) of peribranchial cavity.

oe . Oesophagus.

0. r. "Organe refringent."

pc. Pericardium.

pt. Double-v/alled partition of stolon.

r.a.ex. Right anterior extension or pouch of peribranchial
cavi ty .

r .pbr . c . Right peribranchial cavity.

r.p.ex. Right posterior extension or pouch (right epicar-
dial sac?) of peribranchial cavity.

r.v/.i.v. Thickened wall of inner vesicle on right side.

rd.d.t. Rudiment of dorsal tube.

rd.g. Rudiment of sexual organs.

r d . gl . Rudiment of ganglion.

rd . pc . Rudiment of pericardium.

'1-

St. stomach.

_stol. Stolon.

t. Cellulose test.

•72-

Fig. 1. Total preparaticn of very young bud, shov;ing evagi-
nation of ectodern and partition of stolon. B. 4.

Fig. 2. Slightly older bud, seen from right side. 3. 4.

Fig. 3. Still older bud, seen from right side, and shov/ing
anterior elongation and constriction of the stolon-
ic connection. B. 4.

Fig. 4. Bud, seen from right side, at a stage when devel-
opment of peribranchial cavity, pericardium and di-
gestive tract is well advanced. B. 4.

Fig. 5. Bud, considerably older than last, shov/ing exten-
sions of peribranchial cavity, formation of gill-
slits, and further development of digestive tract.
B. 4.

Fig. 5. Transverse section of stolen, showing double-v-'alled
partition. F. 2.

Fig. 7. Transverse section of stolon at point v:here a bud
is beginning to form. The ectoderm on one side
is thickened, and the double-walled partition is
much thickened and dilated. F. 2.

Fig. 8. Transverse section through very young bud and sto-
lon, at about the stage of Fig. 1. The' walls of
the partition are coming togetner to close off the
inner vesicle. "'esodermal cells are found lying
against the outer surface of the latter. ?. 2.

Fig. 9. Transverse section through posterior end of bud at
about the stage of Fig. 3, showing thicke^ned v;all
of inner vesicle on right side, and beginning of

pericardial rudiiBent . P. 2.

Pig. 10. Transverse section through posterior end of bud,

showing an early stage in the displacernent or shift-
ing of the inner vesicle. The wall of the latter
is being bent in at the point indicated by tne line
a. D. 2.

Pig. 11. Transverse section through posterior end of bud

slightly older than last, showing further progress
of the displacement of the vesicle. The peribran-
chial rudiment is at a much lower level than in the
last figure, and the wall of inner vesicle is being
bent in further at a_. D. 2.

Pig. 12. Transverse section through the middle region of

same bud as the one shov/n in Pig. 11. The section
lies in front of the connection with the stolonic
partition, and shov/s the beginning of the fold
which will form the right peribranchial cavity.
D. 2.

Pigs. 13-18. Series of transverse sections of a bud at

about the same stage as that shown in Pig. 4. "^he
sections are taken respectively at the levels in-
dicated by the lines, a, _b, £, d, _e and f, in Pig.
4. Pig. 1-', line a, the most anterior secticn,
shows undivided portion of branchial sac, and dor-
sal tube. "Pig. 14, line b, is a little furtner
back and shov/s anterior extension of right peri-
branchial cavity. Pig. 15, line _c, is taken imme-

•74-

dieitely in front of the clcacal cavity, and snows
the extensions of tne peribranchial cavity on both
sides. The section passes through the extreme
posterior end of the dorsal tube. Fig. 16, line

d, passes through the anterior ena of the cloacal
cavity, '/nich is seen to con?;oct the lateral por-
tions of the peribrancnial cavity. Fi^:. l^, line

e, is taken from the posterior end of the bud, and
shows the connection of the left peribranchial cav-
ity v^ith the stolonic partition. The pericardial-
sac is also shown in this region. Fig. lo, line

f, snows the undivided extreme posterior end of the
branchial sac. The section passes througn tno ai-
lated portion of the digestivF tract, the stomach,
and behind the connection '-rith the stolonic parti-
tion. All are drawn with D, 2.

Figs. 19-21. Series of transverse sections of a bud of

about the same age as the one represented in Fig.
5. Fig. 19, is a section through the anterior re-
gion, and shov/s the extensions of the peribranchial
cavity. Fig. 20, is dravm from a section .vhich is
taken from the middle region of the bud, and shows
the median portion or cloaca connecting the lateral
sacs of the peribranchial cavity, which is nov/ en-
tirely cut off from, the branchial sac. Fig. 21,
sho\'/s the posterior extensions (epicardial sacs?)
of the peribranchial cavity, and the remnant of the

■75-

connection v/ith the stolcnic partition. D. 2.

Pigs. 22, a and b. These sections illustrate the develop-
ment of the .^ill-slits . Tn a, tiie thickened bran-
chial wall is slightly cvaginated and is in contact
with the thickened patch of cells in the visceral
wall of the peribranchial cavity. The lov/er por-
tion of h^ shov/s the fusion between the two walls,
and the upper portion, a stage after the formation
of the opening. P, 2.

Pigs. 23, a and b^. Stages in the formation of the branchial
orifice. In a, the much-thickened invaginated ec-
toderm is seen nearly touching the branchial v/all.
The fusion of the two walls is shov/n in _b. P. 2.

Pig. 24. Transverse section through the extreme posterior
end of a bud, showing the origin of the gut, as a
diverticulum on the left side of the branchial sac.
D. 2.

Pig. 25. Section through the wall of the stomach and a por-
tion of the duct of the "organe refringent", snov/-
ing clearly the connection of the latter with the
digestive tract. ?. 2.

Pig. 26. Transverse section of the terminal portion of the

. intestine, soov/ing the surrounding ducts and ampul-
lae of the "organe refringent" with tneir deeply
stained nuclei. 7. 2.

Pig. 27, a, b, c^, d, and e_^ Sections illustrating the devel-
opment of the pericardium. i is from a frontal

section of a very young bud, ind sho'-7s tne rudiment
wrien it consists merely of a few cells loosely at-
tached to the inner vesicle. The remaining fig-
ures are drav/n from transverse sections. In h and
c, the rudiment has increased in size, cells are
being added from without, and nuclei v/ithin the
mass are seen to be dividing. Tn d and e^, a cav-
ity has appeared in the centre of t}ie rudiment,
which is now much larger, and the cells are becom-
ing arranged into a one-layered epithelium. .■' . 2.

Fig. 28. Transverse section through tne anterior end of a
biid slightly older than Pig. 9. The rudiment of
the dorsal tube is shown, and consists at this
stage of a few scattered cells, adhering to the
wall of the inner vesicle a little to the left of
the median dorsal line. Tne difference in thick-
ness between the right side and the rest of tne ves-
icle is seen to be but slight in t.iis region. D.2.

Fig. 29, a, b, c, d, and_e . Transverse sections representing
stages in the development of the dorsal tube. In
a a fevf cells are seen loosely grouped together and
lying on trie wall of the inner vesicle. In b^ the
association witn surrounding mesodermal cells is
apparent. In c and d the mass has become compact,
and cell-boundaries have entirely disappeared; in
the latter figure the I'omen in the centre has begun
to form. In e tne tube is completely formed, and

-77-

the cells are arranged around the lumen in a one-
layered epithelium. i'^ . 2.

Fig. 30. Median sagittal section of a bud at about the stage
of Pig. 4, shov/in;':^ the position and extent of tne
dorsal tub«^ . ""ne peribranchial cavity is not yet
completely constricted off from tne branchial sac.
j;. 2.
7ii~. ^1 -_'^i_b,_c,_d , and _e . Transverse section shov/ing the
devrlopment of the ganglion. Tn a and _b a few
cells resembling . lesodermal cells are seen lying
* on the dorsal side of tne dorsal tube. The wall
of the latter is broken at this point, and nuclei
appear to be wandering out into the rudiment . In
£ and _d the rudiment is much enlarged, and in tne
latter trie nuclei are beginning to arrange tnera-
selves in peripheral region vf tne rudiment. In
_e the wall of the dorsal tube under the ganglion
has been reformed, and the central portion of tne
latter is now free from nuclei and occupied by fine
fibrils. P. 2.

Figs. 32, £, b, and c_. Sections illustrating the early de-
velopment of tne sexual organs. Tn a an early
stage is shovm, when the rudiment is merely a loose
mass of cells, surrounding a slight cavity. A
couple of tne elongated cells v/hich will form the
genital cord are also ser-n. In b the cavity of
the rudiment is much enlarged, and the genital cord

is prominent, and attiched to tne sphere; in both
these figures the genital rudiment is seen to lie
close to the wall of t;ie digestive tract. In c
an older stage is shov/n, in which the cavity of the
sphere has become divided into two; tne genital
cord, although only represented in part, now ex-
tends nearly to the posterior wall of tne cloaca,
and lies close to, and parallel with, the intes-
tine. 7, 2.

-79-

VI T A.

The writer of tnis paper, George Lefevre, v/as born
in the city of Balti'nore, "Id., on Septe:nber 16th, 1869.

He received his early education in private, schools
of Balti-nore, and in the fall of 1888, entered the Johns Hop-
kins University as an undergraduate student in the Chemical-
Biological Course.

After taking the degree of Bachelor of Arts in the
same University, he began grad.iate studies in tne Biological
Department, making Zoology his principal subject, and Animal
Physiology and Botany his first and second subordinate sub-
jects, respectively.

In 1892-93, he was assistant in Histology, and the
same year held the University Scholarsnip in Biology.

In 1893-4, he v/as again assistant in Histology;
ir June, 1894, he v/as appointed Fellow in Zoology, and in
June, 1895, received the Bruce Fellowship.

■80-

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