204 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 
then, it is doubly so now, for the literature of the subject has since that 
time multiplied many fold. Nevertheless there still remain many un- 
settled questions regarding the embryology of the Tunicates. Concern- 
ing so fundamental a point as the derivation of the primary germ layers 
in the embryo, quite contradictory opinions have been expressed within 
the last ten years by observers of world-wide reputation. 
I undertook the inquiry, the results of which are recorded in the fol- 
lowing pages, in the hope of being able to throw light on this disputed 
question by the study of other forms than those which had been most 
carefully examined, and by the application of new methods to the prob- 
lem. A short experience convinced me that the only method which could 
yield positive conclusions was that of cell lineage, a method which has 
been applied so successfully to the study of annelid and molluscan em- 
bryology by a number of observers, and had already been employed to a 
limited extent in the study of ascidian embryology by Van Beneden et 
Julin (84), Seeliger (’85), and Chabry (787). 
It soon became clear to me that some of the conflicting statements 
made by my predecessors arose from errors on their part due to incor- 
rect orientation of certain stages. The nature of these errors I have 
fully explained in a preliminary communication (Castle, ’94). A further 
study of the embryonic history, cell by cell, through the periods of cleav- 
age and gastrulation, and even down to the differentiation of the several 
larval organs, has led me to conclusions somewhat at variance with those 
of earlier investigators regarding the origin of the primary germ layers 
and the organs derived from them. One of the most important of these 
conclusions is that the mesoderm of Ascidians — and probably also that 
of Amphioxus and the Vertebrates — is derived in part from the primary 
entoderm and in part from the primary ectoderm. The grounds on 
which this conclusion rests are set forth in the later portions of this 
paper; in the earlier part of the paper I have recorded some observa- 
tions on the maturation and fertilization of the ascidian egg. 
It gives me pleasure to acknowledge in this place my very great obli- 
gations to Professor E. L. Mark for direction and kindly criticism of my 
entire work. My best thanks are also due to Dr. Alexander Agassiz, in 
whose laboratory at Newport the material for my studies was chiefly 
collected, and to Colonel Marshall McDonald for numerous courtesies 
extended to me at the United States Fish Commission Station at 
Wood's Holl, 
