But even granting that there is an historical, embryonic[102] connection, its small importance for the scientific problems connected with embryonic development, and budding and regeneration has been shown by a number of recent discoveries, and nowhere more clearly than in the cases of the formation of new individuals by budding. As an example may be cited the method of development of the ascidian from the egg, and by means of buds. The work of Kowalevsky, Della Valle, Seeliger, and Van Beneden on the budding process of ascidians showed that there are some discrepancies between the bud development and the embryonic development. The more recent papers of Hjort, Oka, Pizon, Salensky, Lefevre, and others have shown very clearly that the germ-layer theory is inapplicable to the bud development in this group. The bud arises as a double-walled tube, or rather a tube within a tube, with a space between. The outer tube comes in all cases from the ectoderm of the animal; the inner tube has a different origin in different species. In perophora, didemnum, and clavellina, the inner tube comes from endoderm; in botryllus it arises from the ectoderm of the larval peribranchial or atrial cavity. In all these forms the inner tube gives rise to the new pharyngeal cavity of the bud, while this same cavity comes from the endoderm of the archenteron of the embryo. In the bud embryo the peribranchial space is also derived from the inner tube; hence it is endodermal in the first series, and ectodermal in botryllus. In the egg embryo it is ectodermal. In regard to the development of the nervous system there is some difference of opinion. A number of investigators have found that the new brain arises from the outer part of the inner or branchial tube, which has in most cases an endodermal origin. Seeliger and Lefevre believe the nervous system to arise from mesodermal cells that lie between the two tubes. It appears, nevertheless, that in several forms the brain really comes from the inner tube, which also gives rise to the branchial sac. Therefore, in those cases in which the inner tube is endodermal the brain has the same origin, and in the case in which the inner tube is ectodermal, the brain is ectodermal, but the pharyngeal sac has also an ectodermal origin. There is obviously no definite relation between the origin of these structures in the bud and in the egg embryo. A similar difficulty is met with in the Bryozoa in regard to the development of the egg embryo and the bud embryo.