Chapter III: Introduction: Outline of History—structure of a Typical Ascidian—embryology (1)

AND LIFE-HISTORY

The TUNICATA are marine animals found in practically all parts of the sea, and at all depths. They extend from the Arctic and Antarctic regions to the tropical waters, and from the littoral zone down to the abyssal depths of over three miles. They are abundant in British seas. They vary greatly in shape and colour, and range in size from an almost invisible hundredth of an inch to large masses a foot or more in diameter. And yet most Tunicata have a characteristic appearance by which they can be readily distinguished from other animals. They form a well-defined group, with definite anatomical characters, and there are no known forms intermediate between them and other groups. The Tunicata were formerly regarded as constituting, along with the Polyzoa and the Brachiopoda, the Invertebrate Class "MOLLUSCOIDEA." They are now known to be a degenerate branch of the lower CHORDATA, and to be more nearly related to the Vertebrata than to any group of Invertebrates.

Tunicata occur either fixed or free, solitary, aggregated or in colonies (see Fig. 27, p. 64). The fixed forms, found on the sea-bottom, are usually termed "Ascidians," those that are solitary or merely aggregated being "Simple Ascidians" or Monascidiae, and those that are organically united into a colony being "Compound Ascidians" or Synascidiae. The colonies have been produced by budding, a process which is very general in the group, and the members of the colony are conveniently known as "Ascidiozooids." Some exhibit {36}alternation of generations, and all pass through remarkable changes in their life-history, nearly all of them undergoing a retrogressive metamorphosis.

OUTLINE OF HISTORY.

More than two thousand years ago Aristotle gave a short account of a Simple Ascidian under the name of _Tethyum_. He described the appearance and some of the more important points in the anatomy of the animal. From that time onwards comparatively little advance was made until Schlosser and Ellis, in a paper on _Botryllus_, published in the _Philosophical Transactions_ of the Royal Society for 1756, first brought the Compound Ascidians into notice. It was not, however, until the commencement of the nineteenth century, as a result of the careful anatomical investigations of Cuvier[66] upon the Simple Ascidians, and of Savigny[67] upon the Compound Ascidians, that the relationship between these two groups of Tunicata was conclusively demonstrated. Up to 1816, the date of publication of Savigny's great work, the few Compound Ascidians previously known had been generally regarded as Alcyonaria or as Sponges; and although many new Simple Ascidians had been described by O. F. Müller[68] and others, their internal structure had not been investigated. Lamarck[69] in 1816, chiefly as the result of the anatomical discoveries of Savigny and Cuvier, instituted the class TUNICATA, which he placed between the Radiata and the Vermes in his system of classification. The Tunicata included at that time, besides the Simple and the Compound Ascidians, the pelagic forms _Pyrosoma_, which had been first made known by Péron in 1804, and _Salpa_ described by Forskål in 1775.

Chamisso, in 1819, made the important discovery that _Salpa_ in its life-history passes through the series of changes which were afterwards more fully described by Steenstrup in 1842 as "alternation of generations"; and a few years later Kuhl and Van Hasselt's investigations upon the same animal resulted in the discovery of the alternation in the directions in which the wave of contraction passes along the heart, and in which the {37}blood circulates through the body. It has since been found that this observation holds good for all groups of the Tunicata. In 1826, H. Milne-Edwards[70] and Audouin made a series of observations on living Compound Ascidians, and amongst other discoveries they found the free-swimming tailed larva and traced its development into the young Ascidian.

In 1845, Carl Schmidt[71] first announced the presence in the test of some Ascidians of "tunicine," a substance very similar to cellulose; and in the following year Löwig and Kölliker[72] confirmed the discovery, and made some additional observations upon this substance and upon the structure of the test in general. Huxley,[73] in an important series of papers published in the _Transactions_ of the Royal and Linnean Societies of London from 1851 onwards, discussed the structure, embryology, and affinities of the pelagic Tunicates, _Pyrosoma_, _Salpa_, _Doliolum_ and _Appendicularia_. These important forms were also investigated about the same time by Gegenbaur, Vogt, H. Müller, Krohn, and Leuckart.

The most important epoch in the history of the Tunicata is the date of the publication of Kowalevsky's celebrated memoir[74] upon the development of a Simple Ascidian. The tailed larva had been previously discovered and investigated by several naturalists, notably by H. Milne-Edwards,[75] P. J. van Beneden, and Krohn; but its minute structure had not been sufficiently examined, and the meaning of what was known of it had not been understood. It was reserved for Kowalevsky in 1866 to demonstrate the striking similarity in structure and in development between the larval Ascidian and the Vertebrate embryo. He showed that the relations between the nervous system, the notochord, and the alimentary canal are practically the same in the two forms, and have been brought about by a very similar course of embryonic development. This discovery clearly indicated that the Tunicata are closely allied to Amphioxus and the Vertebrata, and that the tailed larva represents the primitive or ancestral form from which the adult Ascidian has been evolved by degeneration. This led naturally to the view usually accepted at the present day, that the group is a degenerate {38}side-branch from the lower end of the phylum CHORDATA, which includes the Tunicata (or Urochordata), Balanoglossus and its allies (Hemichordata), Amphioxus (Cephalochordata), and the Vertebrata (or Craniata). Kowalevsky's great discovery has since been confirmed and extended to all other groups of the Tunicata by Kupffer,[76] Giard, and others.

In 1872 Fol[77] added largely to the knowledge of the Appendiculariidae, and Giard[78] to that of the Compound Ascidians. The latter author described a number of new forms and remodelled the classification of the group. The most important additions which have been made to the Compound Ascidians since Giard's work have been the species described by von Drasche,[79] from the Adriatic, and those discovered by the "Challenger" expedition.[80] The structure and the systematic arrangement of the Simple Ascidians have been discussed of recent years mainly by Alder[81] and Hancock, Heller,[82] Lacaze-Duthiers,[83] Traustedt,[84] Roule, Hartmeyer, Sluiter[85] and Herdman.[86] In 1874 Ussoff investigated the minute structure of the nervous system and of the underlying gland, which was first discovered by Hancock, and showed that the gland has a duct which communicates with the front of the branchial sac or pharynx by an aperture in the dorsal (or "olfactory") tubercle. In an important paper published in 1880, Julin[87] drew attention to the similarity in structure and relations between this gland and the "hypophysis cerebri" of the Vertebrate brain, and insisted upon their homology. Metcalf has recently added further to our knowledge on this and related matters.

The Thaliacea or pelagic Tunicata have of late years been the subject of several very important memoirs. The researches {39}of Todaro, Brooks,[88] Salensky,[89] Seeliger,[90] Korotneff,[91] and others have elucidated the embryology, the gemmation and the life-history of the Salpidae; and Grobben, Barrois,[92] and more especially Uljanin,[93] have elaborately worked out the structure and the details of the complicated life-history of the Doliolidae. Finally we owe to the labours of Metschnikoff, Kowalevsky, Giard, Hjort, Seeliger, Ritter, Van Beneden and Julin, much detailed information as to development and life-history, the process of gemmation and the formation of colonies, which has added greatly to our knowledge of the position and affinities of the Tunicata and of their natural classification.

STRUCTURE OF A TYPICAL ASCIDIAN.

If a typical "Simple Ascidian," such as the common British _Ascidia mentula_ (Fig. 15), or _Ascidia virginea_, be examined alive and expanded in sea-water it will be seen to bear on the upper surface two short projections, each terminated by a wide tubular opening, through which the animal, when touched, can emit jets of water with considerable force—thus accounting for the popular name "sea-squirts." The rest of the body is covered by the dull grey tough cuticular outer "test" or "tunic" (hence TUNICATA) by means of which the animal is attached to a rock or other foreign body. One of the tubular openings, the mouth or "branchial aperture," is terminal, and indicates the morphological anterior end; it is surrounded by eight lobes. The other opening, the cloaca or "atrial aperture," is on the dorsal edge, from one-third to one-half way down the body, and is bounded by six lobes only; consequently the two apertures, and so the ends of the body, can be distinguished externally by the number of lobes—an important matter. The area of attachment is usually the posterior part of the left side; in Fig. 15 the animal is seen from the right hand side.

If a little carmine-powder, or some other insoluble particles be scattered in the water in which the Ascidian is living, the {40}particles will be seen to converge to the branchial aperture and be sucked in by the inhalent current entering the body. After a short interval a certain proportion of the particles will be shot out from the atrial aperture with the exhalent current.

These particles have passed through the pharyngeal portion of the alimentary canal and the cloacal passages, with the water used in respiration, but a considerable amount of such particles taken in with the water do not reappear, as they are retained by the nutritive organs and pass along the remainder of the alimentary canal with the food. The current of water passing in at the branchial and out at the atrial aperture is of primary importance in the life of the Ascidian. Besides serving for respiratory purposes it conveys all the food into the body and removes waste matters both intestinal and renal, and also expels the reproductive products from the body.

THE TEST.—The test is notable amongst animal structures for containing "tunicine," a substance which appears to be identical in composition, and in behaviour under treatment with various reagents, with cellulose. It is cartilaginous in appearance and consistency, and to some extent in structure, as it consists of a clear (or in some cases fibrillated) matrix in which are embedded many corpuscles or cells. It is the matrix that contains the cellulose, which may form over sixty per cent by weight of the entire test. As the test is morphologically a cuticle, being a secretion on the outer surface of the ectoderm (Fig. 16, _ec_), the cells it contains have immigrated to it from the body, and it has recently been shown that many of these are mesodermal cells (leucocytes or connective tissue wandering cells, amoebocytes, and in some cases embryonic "kalymmocytes," or {41}egg-follicle cells, see below, p. 56), which have passed through the ectoderm. This process commences in the larval state with the migration of mesenchyme cells from the blastocoele through the epiblast. Ectoderm cells, and possibly also some primitive endoderm cells, also take part in forming the test. Many of these cells in the test remain small and simple, as the fusiform and stellate test-cells; some become pigment-cells, while others enlarge and become vacuolated to form the large (up to 0.15 mm. in diameter) vesicular or "bladder" cells—this is especially the case in the outer layer of the test in _Ascidia mentula_ (see Fig. 17, _bl_) where there are innumerable clear vesicles, each surrounded by a thin film of protoplasm and having the nucleus still visible at one point of the surface. In some of the Tunicata the test-cells produce calcareous spicules of various shapes (see below, p. p. 86).

The test also becomes organised by the growth into it of the so-called "vessels." These are outgrowths of the body-wall covered by ectoderm and containing prolongations of blood-channels from the connective tissue of the "mantle" (body-wall). Fig. 16, _t.v_ shows such an outgrowth, and exhibits the general relations of test (cuticle), ectoderm, and mesoderm. It also explains how it is that the blood-channel being pushed out as a {42}loop gives rise to the double or paired "vessels" seen branching through the test (see Fig. 17, _v_). The two vessels of a pair are one blood-channel imperfectly divided by a connective-tissue septum. The blood courses out along one side, round the communication in a "terminal knob" at the end, and back down the other side. The "terminal knobs" are very numerous, and form a marked feature in the outer layer of the test (Fig. 17, _t.k_); in some cases (_Culeolus murrayi_), they probably form an accessory organ of respiration, while in others (Botryllidae), they pulsate and aid in keeping up the circulation.

The ectoderm is a simple epithelial layer (Fig. 16, _ec_). It is turned in for a short distance at the branchial aperture (mouth), and atrial aperture (cloaca), as a short stomodaeum and proctodaeum respectively, lined in each case by a delicate prolongation of the test.

Fig. 24, A, p. 52, shows the relations of ectoderm, mesoderm, and endoderm in a section through the antero-dorsal part of the body. The cavity marked _p.br_ is a portion of the atrial cavity lined by ectoderm, and must not be confounded with a coelom. The absence of a true coelom in the mesoderm will be noticed in this and other figures, and yet the Tunicata are Coelomata, although it is very doubtful whether the enterocoel which has been described in the development of some is ever found. The coelom is in any case largely suppressed later, and is only represented in the adult by the pericardium and by small cavities in the renal and reproductive organs and ducts.

BODY-WALL AND CAVITIES OF THE BODY.—The name "mantle" is given to the ectoderm with the parietal mesoderm which form the body-wall inside the test. It is largely formed of connective {43}tissues—both homogeneous and fibrous—with cells, blood-sinuses, and many muscle-bundles large and small running circularly, longitudinally, and obliquely, and interlacing in all directions (Fig. 18, _m_). The muscles are all formed of very long fusiform non-striped fibres. The mantle in some Ascidians is often brilliantly pigmented—red, yellow and opaque white, the coloured cells being exactly like those found in the blood.

The mantle forms two well-marked siphons or short wide tubes, which lead in from the branchial and atrial apertures. These are surrounded by strong sphincter muscles,[94] and are lined by the invaginated ectoderm and test. The one leads into the branchial sac or modified pharynx, and the other into the atrial or peribranchial cavity (see Fig. 18, and Fig. 19, _p.br_).

Figs. 18 and 19 show the relations of the branchial and peribranchial cavities to one another. The peribranchial cavity {44}opens to the exterior dorsally by the atrial aperture, forms the cloaca along the dorsal edge of the body, and has extensions laterally on each side of the branchial sac, with the interior of which it is placed in communication by the secondary gill-slits or "stigmata" (Fig 19, _sg_). Along the ventral edge the mantle is united to the wall of the branchial sac, and it is only this union (Fig. 19, _end_) that prevents the peribranchial cavity from completely surrounding the branchial sac.

The following list of the cavities present in the body of the adult _Ascidia_ may be useful at this point:—

1. The alimentary canal, including the branchial sac. This is derived from the archenteron of the embryo, is lined throughout by endoderm, and the system of cavities of the intestinal gland is to be regarded merely as an outgrowth from the alimentary canal.

2. The peribranchial (atrial) cavity, derived from two lateral ectodermal invaginations which join dorsally to form the cloaca and open to the exterior by the atrial aperture.

3. The original embryonic segmentation cavity (blastocoele) remains, where not obliterated by the development of the mesodermal connective tissue, as the irregular system of blood spaces, with its outgrowths in test and branchial sac. The heart, which has differentiated muscular walls, becomes secondarily connected at its ends with these blood spaces.

4. The pericardium and epicardium (see p. 83) originate as outgrowths from the archenteron. They may therefore be regarded as enterocoelic spaces. The pericardium becomes completely closed off and separated from the alimentary canal. The epicardium may form paired tubes of great length, and may remain permanently connected with the branchial sac.

5. The cavities of the renal vesicles and of the gonads and ducts are spaces formed in the mesoblast. They have been variously interpreted:—

(_a_) As of the same nature as the blood spaces (blastocoelic), or

(_b_) As formed by a splitting of the mesoblast (coelomic).

6. The cavity of the neural gland and of its duct opening at the dorsal tubercle is derived from the primitive dorsal neural tube of the embryo, and so may be regarded as a part of the lumen of the cerebro-spinal nervous system.

TENTACLES, ETC.—The branchial aperture leads through the {45}branchial siphon into the branchial sac. At the base of the siphon, just about the line of junction of the ectoderm of the stomodaeum with the endoderm of the mesenteron, is placed a circle of simple hair-like tentacles (Fig. 18, _tn_) which stand out at right angles to the wall, and more or less completely meet in the centre to form a delicate, sensory grid or sieve through which all the water entering the body has to pass. These tentacles not only act mechanically, but are also sensitive although only scattered sensory cells, and no specially differentiated sense-organs are found upon them. Behind the tentacles lies the plain, or papillated, prebranchial zone (Fig. 21, _p.br.z_), bounded behind by a pair of parallel and closely placed ciliated ridges with a groove between—the peripharyngeal bands—which encircle the anterior end of the branchial sac.

The branchial sac is very large—much the largest organ of the body—and extends almost to the posterior end of the body, while the rest of the alimentary canal lies upon its left side. The food particles, consisting of microscopic plants and animals, are carried in through the branchial aperture by the current of water, but most of them do not pass out through the gill-slits to the atrium, being entangled in the viscid mucus which passes by ciliary action along the groove between the peripharyngeal bands.

{46}ENDOSTYLE.—The mucus just referred to is produced in the long canal-shaped gland called the endostyle or hypobranchial groove, which runs along the entire ventral edge of the branchial sac (Fig. 18, _end_). The sides, and especially the floor of the endostyle, are richly ciliated, while there are four (or six) strongly-marked, peculiarly-shaped glandular tracts, two (or three) on each side (Fig. 20, _gl_) running along its length, and separated by areas of closely-packed fusiform cells with short cilia, amongst which are found some bipolar sensory cells.

This organ corresponds to the hypopharyngeal groove of Amphioxus and the median part of the thyroid gland of Vertebrata. It is interesting to notice that the (at least) four longitudinal tracts of gland-cells are of remarkable constancy, being found not only in all groups of Tunicata, including even the pelagic, tailed Appendicularians, but also in Amphioxus and in the young thyroid gland of the Ammocoete. When, in Ascidians, a third marginal glandular tract is added it has a different appearance from the two characteristic tracts. The mucus is carried forward by the action of the large floor-cilia of the endostyle (Fig. 20) to the groove between the peripharyngeal bands, and after encircling the anterior end of the branchial sac and collecting the food particles, it passes backwards along the dorsal edge {47}of the branchial sac to the oesophagus, guided by a membranous fold, the dorsal lamina (Fig. 21, _d.l_), which is more or less ridged or corrugated, and may be armed with marginal tags or even replaced by larger processes (the "languets") in some species of Ascidians. In the living animal the lamina has its free edge curved to the right hand side in such a manner as to constitute a fairly perfect tube along which the train of food passes.

BRANCHIAL SAC.—Thus we have the dorsal lamina (or the languets) along the dorsal edge, the endostyle along the ventral edge, and the peripharyngeal bands around the anterior end. The wall of the branchial sac itself is penetrated by a large number of channels through which blood flows. Some of these run in one direction and some in another, so as to form complicated networks, which differ greatly in their arrangement in different Ascidians. Between these blood-channels there are clefts ("stigmata"), the secondary or subdivided gill-slits, by means of which the current of water passes from the branchial sac to the large external peribranchial or atrial cavity. All the stigmata (of which there may be several hundred thousand) in the wall of the branchial sac are bounded by cubical or columnar epithelial cells, which are ciliated. These cilia, so long as the animal is alive, are in constant motion, so as to drive the water onwards, and it is this constant ciliary action in the walls of the branchial sac that gives rise to the all-important current of water streaming through the body. In addition to the stigmata there are generally one or two much larger elongated slits (Garstang's {48}pharyngo-cloacal slits) placed close to the dorsal lamina and leading direct to the cloaca.

Fig. 22 shows a small part of the wall of the branchial sac, in which it may be seen that the bars containing the blood-channels are arranged in three regular series:—(1) The "transverse vessels" which run horizontally round the wall and open at their dorsal and ventral ends into large median longitudinally running tubes, the dorsal blood-sinus (or "dorsal aorta") behind the dorsal lamina, and the ventral blood-sinus (or "branchial aorta") beneath the endostyle; (2) the fine longitudinal or "interstigmatic vessels" which run vertically between adjacent transverse vessels and open into them, and which therefore bound the stigmata; and (3) the "internal longitudinal bars" which run vertically, in a plane internal to that of the transverse and fine longitudinal vessels. These bars (Fig. 22, _i.l_) communicate with the transverse vessels by short side branches where they cross, and at these points are prolonged into the cavity of the sac in the form of hollow papillae. In some Ascidians (e.g. _Corella_ and most of the Molgulidae) the interstigmatic vessels are curved so that the stigmata form more or less complete spirals (see Figs. 35 and 41). In some species of _Ascidia_, and other Ascidians, the interstigmatic vessels are inserted into the transverse vessel in an undulating course in place of the straight line seen in Fig. 22, B, _l.v_, the result being that the stigmatic part of the wall of the branchial sac seems to be folded or thrown into microscopic crests and troughs. This is known as "minute plication." In some cases, again (Cynthiidae), the whole wall of the sac is pushed inwards at intervals to form large folds visible to the eye (see Fig. 36, A and B). The intersections of the internal longitudinal bars with the transverse vessels divide up the inner surface of the branchial {49}sac wall into rectangular areas called "meshes." One such mesh, containing eight stigmata in a row, is seen in Fig. 22, A. The internal longitudinal bars bear papillae at the angles of the meshes, and occasionally in intermediate positions. There are frequently horizontal membranes (Fig. 22, B, _h.m_) attached to the transverse vessels between the papillae. There are many "connectives" running from the outer wall of the branchial sac to the mantle outside, and allowing the blood in the transverse vessels to communicate with that in the sinuses of the mantle (see Fig. 19, _con_).

HEART AND CIRCULATION.—It is one of the notable features of the Tunicata that the circulation is not constant in direction, but is periodically reversed.

The blood of Ascidians is in the main transparent, but usually contains certain pigmented corpuscles in addition to many ordinary leucocytes or colourless amoeboid cells. The pigment in the coloured cells may be red, yellow, brown, or in some cases blue or opaque white. The blood may reach the branchial sac either from the dorsal or from the ventral median sinus according to the direction in which the heart is beating at the moment (see below); and it is a most interesting and beautiful sight to see the circulation of the variously coloured corpuscles through the transparent vessels, and the lashing of the cilia along the edges of the neighbouring stigmata in a small Ascidian under the microscope.

In _Ascidia_ (Fig. 23) the heart is an elongated fusiform tube placed on the ventral and posterior edge of the stomach, projecting into a space (the pericardium) which is a part of the original coelom, the remainder of which is represented in the adult by the reproductive and renal cavities. The wall of the heart is continuous along one edge with that of the pericardium, and the heart is to be regarded as a tubular invagination of the pericardial wall, shutting in a portion of the surrounding space (the blastocoel of the embryo), and having open ends which communicate with the large blood sinuses leading to the branchial sac, to the viscera, and to the body-wall and test. The cavity of the heart is not divided and there are no valves. Its wall is formed of a single layer of epithelio-muscular cells, the inner, muscular, ends of which are cross-striated fibres running round the heart—the only striated muscular tissue found in the body. Waves of {50}contraction pass along the heart from end to end, first for a certain number of beats in one direction, and then, after an interval, in the other. If a small or young _Ascidia_ be placed alive, left side uppermost, in a watch-glass or small trough of sea-water, and examined with a low power of the microscope, the heart will be readily seen near the posterior end of the transparent body. It will be noticed that the "beating" looks like successive waves of blood pressed through the tubular heart from one end to the other by its contractions. After watching the waves passing, let us say, from the right hand end of the heart to the left for about a minute and a half (perhaps 60 or 80 to 100 beats), it will be seen that they gradually become slower and then stop altogether. But after seven or eight seconds a faint wave of contraction will start from the _left_ end of the heart and pass over it to the right; and this will be followed by larger ones for a minute and a half, and then again a pause will occur and the direction change. It has been suggested that the cause of this remarkable reversal may possibly be that the heart being on the ventral vessel, which is wider than the corresponding dorsal trunk, pumps the blood into either the lacunae of the branchial sac or those of the viscera in greater volume than can possibly get out through the smaller branchio-visceral vessel in the same time, the result being that the lacunae in question soon become engorged, and by back pressure cause the stoppage, and then reversal of the beat. The absence of any valves in the heart to regulate the direction of flow obviously facilitates this alternation of the current.

The larger channels through which the blood flows may be lined with a delicate endothelium, but the smaller passages are merely spaces in the connective tissue. The heart, although anatomically a "ventral vessel," runs in the main dorso-ventrally. The blood-channel leaving the ventral end of the heart is the "branchio-cardiac vessel" (Fig. 23, _b.c_). This gives off a branch which, along with a corresponding branch from the "cardio-visceral" vessel (_c.v_) at the other end of the heart, goes to the test, and then runs along the ventral edge of the branchial sac as the branchial aorta (_b.a_), external to the endostyle, communicating laterally with the ventral ends of all the transverse vessels of the branchial sac. The cardio-visceral vessel (Fig. 23, _c.v_) after giving off its branch to the test breaks up into a number of sinuses which {51}ramify over the alimentary canal and the other viscera. These visceral lacunae finally communicate with a third great sinus, the "branchio-visceral" vessel (_b.v_) which runs forward along the dorsal edge of the branchial sac as the dorsal aorta (_d.a_), externally to the dorsal lamina, and joins the dorsal ends of all the transverse vessels of the branchial sac. Besides these three chief systems—the branchio-cardiac, the cardio-visceral, and the branchio-visceral—(see Fig. 23), there are numerous lacunae in all parts of the body by means of which anastomoses are established between the different currents of blood.

When the heart contracts ventro-dorsally the course of the circulation is as follows:—the blood which is flowing through the vessels of the branchial sac is collected in an oxygenated condition in the branchio-cardiac vessel, and after receiving a stream of blood from the test enters the ventral end of the heart. It is then propelled from the dorsal end into the cardio-visceral vessels, and so reaches the test and the digestive and other viscera; then, after circulating in the visceral lacunae it passes into the branchio-visceral vessel in an impure condition, and is distributed to the branchial vessels to be purified again. When the heart, on the other hand, contracts dorso-ventrally, this course of the circulation is reversed, the "veins" and "arteries" exchange functions, and what a minute before was a "systemic," is now a "respiratory" heart. This is a phenomenon without parallel in the animal kingdom.

{52}All the blood-spaces and lacunae are probably derived, like the cavity of the heart, from the blastocoel of the embryo, and are not, like the cavity of the pericardium, a part of the coelom (of endodermal origin).

NEURAL GLAND AND DORSAL TUBERCLE.—In the dorsal median line near the anterior end of the body, and imbedded in the mantle on the ventral[95] surface of the nerve-ganglion, there lies a small glandular mass—the neural gland—which, as Julin first showed, there is some reason to regard as the homologue of the hypophysis cerebri of the Vertebrate brain. Metcalf has recently shown that the neural gland may be a double structure—partly cerebral and partly stomodaeal—as in Vertebrates.

The function of this gland is still somewhat mysterious. It may merely form the viscid secretion which is carried along the peripharyngeal bands and down the dorsal lamina. On the other hand, it has been suggested that the function of the organ may possibly be renal, for the removal of nitrogenous waste matters in the neighbourhood of the nervous system. Finally, it may be a lymph gland.

{53}The neural gland, which was first noticed by Hancock, may be continued backwards along with the dorsal nerve, and it communicates anteriorly by means of a narrow duct with the front of the branchial sac (pharynx). The opening of the duct is enlarged to form a funnel-shaped cavity (Fig. 24, A), which may be folded upon itself, convoluted, or even broken up into a number of smaller openings (see Fig. 43, p. 79), so as to form a complicated projection called the dorsal tubercle, situated in the dorsal part of the prebranchial zone. The dorsal tubercle in _Ascidia mentula_ is somewhat horse-shoe shaped (Fig. 21, _d.t_); it varies in most Ascidians (see Fig. 43) according to the genus and species, and in some cases in the individual also. Sensory cells are found in the epithelium, and so it is highly probable that besides being the opening of the duct from the neural gland, this convoluted ciliated ridge may be a sense-organ for testing the quality of the water entering the branchial sac.

NERVOUS SYSTEM AND SENSE-ORGANS.—The single elongated ganglion (Fig. 24, _n.g_), in the median dorsal line of the mantle, between the branchial and atrial siphons, is the only nerve-centre in _Ascidia_ and most other Tunicata. It is the degenerate remains of the dorsal wall of the tubular cerebro-spinal nervous system of the trunk-region of the tailed larval Ascidian—the ventral wall opposite having given rise to the subneural gland. The more posterior or spinal part of the larva has almost entirely disappeared in most adult Tunicata. It persists, however, in the Appendiculariidae, and traces of it have been found in the dorsal nerve running backwards towards the oesophagus in some Ascidians (e.g. _Clavelina_). It may be ganglionated in Molgulidae.

The ganglion has small rounded nerve-cells on its surface, and interlacing nerve-fibres inside. It gives off distributory nerves at both ends (Fig. 24, A), which run through the mantle to the neighbourhood of the apertures, where they divide up to supply the lobes and the sphincter muscles. The only sense-organs are the pigment spots ("ocelli," formed of modified ectoderm cells imbedded in red and yellow pigment), between the branchial and atrial lobes, the tentacles at the base of the branchial siphon, and probably the dorsal tubercle and the languets or dorsal lamina, in all of which, as well as in the endostyle and peripharyngeal bands and in papillae on the ectoderm and in the branchial sac, sensory cells have been found. {54}These, considered as sense-organs, are all in a lowly-developed condition. The larval Ascidians, on the other hand, have well-developed intra-cerebral optic and otic sense-organs (see Fig. 26, p. 60), and in some of the pelagic Tunicata, otocysts and pigment-spots are found in connexion with the ganglion.

ALIMENTARY CANAL.—The mouth and pharynx (branchial sac) have already been described. The remainder of the alimentary canal is a bent tube, which in _A. mentula_ and most other Ascidians lies imbedded in the mantle on the left side of the body, and projects into the peribranchial cavity (see Figs. 18 and 19). The oesophagus leaves the branchial sac in the dorsal middle line, near the posterior end of the dorsal lamina. It is a short curved tube which leads ventrally to the large fusiform thick-walled stomach, ridged internally. The intestine emerges from the ventral end of the stomach and soon turns anteriorly, then dorsally, and then posteriorly, so as to form a curve, the intestinal loop, in which the ovary lies, open posteriorly. The intestine now curves anteriorly again, and from this point runs nearly straight forward as the rectum, thus completing a second curve, the rectal loop, in which the renal vesicles lie, open anteriorly. The wall of the intestine is thickened internally to form the typhlosole (Fig. 18, _ty_), a pad which runs along its entire length, so as to reduce the lumen of the tube to a crescentic slit. The anus opens into the dorsal or cloacal part of the peribranchial cavity near the atrial aperture. The walls of the stomach are glandular, and most of the endoderm cells lining the tube are ciliated. A system of delicate, microscopic, branched tubules with dilated ends (the "refringent organ"), which ramifies over the outer wall of the intestine, and communicates with the cavity of the stomach at the pyloric end by means of a duct is probably a digestive gland. There is in _Ascidia_ no separate large gland to which the name "liver" can be applied, as in some other Tunicata.

RENAL ORGAN.—A mass of large clear-walled vesicles which occupies the rectal loop (Figs. 18 and 19, _ren_), and may extend over the adjacent walls of the intestine, is a renal organ without a duct. Each vesicle is the modified remains of a part of the primitive coelom or body-cavity, and is formed of cells which eliminate nitrogenous waste matters from the blood circulating in the neighbouring blood-lacunae, and deposit them in the cavity of {55}the vesicle, where they form one or more concentrically laminated concretions of a yellowish or brownish colour, sometimes coated with a chalky deposit. These concretions contain uric acid, and in a large Ascidian are very numerous. The nitrogenous waste products are thus deposited and stored up in the renal vesicles in place of being excreted from the body. In other Ascidians the renal organs may differ from the above in position and structure; but in no case have they any excretory duct, unless the neural gland is to be regarded as one of the renal organs—which has not yet been proved.

REPRODUCTIVE ORGANS.—_Ascidia mentula_ is hermaphrodite, and the reproductive organs lie with the alimentary canal, on the left side of the body (Fig. 19, _ov_). The ovary is a ramified gland which occupies the greater part of the intestinal loop. It contains a cavity which, along with the cavities of the testis, is derived from an embryonic coelom; the ova are formed from its walls, and fall when mature into the cavity. The oviduct is continuous with the cavity of the ovary, and leads forward alongside the rectum, finally opening near the anus into the peribranchial cavity (Fig. 18, _g.d_). The testis is composed of a great number of delicate, branched tubules, which ramify over the ovary and the adjacent parts of the intestinal wall. These tubules terminate in ovate swellings. Near the commencement of the rectum the larger tubules unite to form the vas deferens, a tube of considerable size, which runs forward alongside the rectum, and, like the oviduct, terminates by opening into the peribranchial cavity close to the anus. The lumen of the tubules of the testis, like the cavity of the ovary, is a part of the embryonic mesoblastic space, and the spermatozoa are formed from the cells lining the wall. In some Ascidians (certain Molgulidae and Cynthiidae), reproductive organs are present on both sides of the body, and in others, as in _Polycarpa_, there are many complete sets of both male and female systems attached to the inner surface of the mantle on both sides of the body and projecting into the peribranchial cavity.

EMBRYOLOGY AND LIFE-HISTORY OF A TYPICAL ASCIDIAN.

The eggs of Tunicata are for the most part of small size, nearly colourless and transparent, and with little or no food-yolk. {56}In some, however (such as some of the Cynthiidae, and some Compound Ascidians), the eggs are larger, more opaque, and have a fair amount of food-yolk. Ova of this type are not expelled from the body of the parent as ova, but are fertilised, and remain in the atrial cavity or in a special diverticulum thereof—the incubatory pouch—until they are far advanced in development; and usually leave the body as tailed larvae. In many species, the ova and spermatozoa mature at different times in the life-history, and so self-fertilisation is prevented. Some species (such as many Botryllidae and Distomatidae) are protogynous, the ova being produced and shed before the testes have matured, while other species (_Coelocormus huxleyi_) are protandrous, being male while young and female later. But there is no doubt that in other cases (e.g. _Ascidia mentula_) self-fertilisation is not only possible, but does take place. After maturation certain of the follicle-cells which invest the ovum in the ovary migrate into the egg and proliferate so as to form a layer in the superficial part of the egg, where they appear as the so-called "testa-cells" or "kalymmocytes" (Fig. 25, A, _t.c_). The remaining follicle-cells may form two or more layers, usually one of large cubical cells, which may become greatly vacuolated, next to the ovum, and an external flattened layer which is cast off when the egg escapes from the ovary.

Segmentation is complete and results in the formation of a spherical blastula with a small segmentation-cavity (Fig. 25, C). The blastula grows larger and begins to differentiate.[96] There are slightly smaller cells which divide more rapidly at one end of this embryo, the future ectoderm, and slightly larger and more granular cells at the other, which become chiefly endoderm (hypoblast). Invagination of the larger cells then takes place (Fig. 25, D), resulting in the formation of a gastrula with an archenteron. The hypoblast cells lining the archenteron become columnar (_hy_). The curving and more rapid growth at the anterior end of the embryo narrow the primitively wide open blastopore, and carry it to the posterior end of the future dorsal surface (Fig. 25, E). The orientation of the body is now clear.

{57}

The embryo is elongated antero-posteriorly, the dorsal surface is flattened, and the blastopore indicates its posterior end. Around the blastopore the large ectoderm cells form a medullary plate, along which a groove (the medullary groove), runs forwards, bounded at the sides by medullary folds which meet behind the blastopore. Underneath the posterior part of the medullary groove certain of the hypoblast cells from the dorsal wall of the archenteron, in the median line, form a band extending forwards (Fig. 25, E, _ch_). This band separates off from the hypoblast, which closes in beneath it, and thus gives rise to the notochord (Fig. 25, F). The more lateral and posterior cells become mesoblast, and separate off as lateral plates, which show no trace of metameric segmentation (Fig. 25, G). The remainder of the archenteron {58}becomes the branchial sac, and by further growth buds off the rest of the alimentary canal.

The medullary groove now becomes converted into the closed neural canal by the growing up and arching inwards (Fig. 25, G, _n.c_) of the medullary folds, which unite with one another from behind forwards in such a way that the blastopore now opens from the enteron into the floor of the neural canal, forming the neurenteric passage (Fig. 25, F, _n.e.c_). For a time the anterior end of the neural canal remains open as a neuropore. By this time the posterior end is elongating to form a tail, and the embryo is acquiring the tadpole-shape (Fig. 25, H) characteristic of the free larva. The tail grows rapidly, curves round the body, and also undergoes torsion, so that its dorsal surface comes to lie on the left side. It contains ectoderm cells on its surface, notochordal cells (in single file) up the centre (see Fig. 25, H, _ch_), a neural canal dorsally, and a row of endoderm cells representing the enteron ventrally to the notochord. Later on the mesoblast also is prolonged into the tail, where it forms a band of striated muscle-cells at each side of the notochord. When the ectoderm cells begin to secrete the cuticular test this forms two delicate transparent longitudinal (dorsal and ventral) fins in the tail (Fig. 25, K, _f_), and especially at its extremity where radial thickenings form striae resembling fin-rays. The ectoderm on the anterior end of the body grows out into three adhering papillae (Fig. 26, A).

The neural canal now differentiates into a tubular dorsal nervous system. The anterior end dilates to form the thin-walled cerebral vesicle (see Figs. 25, I, and 26, A), containing later the intra-cerebral, dorsal, pigmented eye (_oc_), and the ventral otolith (_au_) of the larva. The next part of the canal thickens to form the trunk-ganglion, and behind that is the more slender "spinal cord," which runs to the extremity of the tail. A ciliated diverticulum of the anterior end of the enteric cavity (future pharynx) which enters into close relations with the front of the cerebral vesicle,[97] and later opens into the ectodermic invagination which forms the mouth at that spot, is evidently the rudiment of the neural duct or hypophysial canal. The future branchial sac (pharynx), with a ventral median thickening which will be the endostyle, is by this time clearly distinguishable by its large size {59}from the much narrower posterior part of the enteron, which grows out to become the oesophagus, stomach, and intestine. The notochord does not extend forward into the pharyngeal region, but is confined to the posterior or caudal part of the embryo. It now shows lenticular pieces of a gelatinous intercellular substance secreted by the cells and lying between them (Fig. 25, I). The mouth forms as a stomodaeum, or ectodermal invagination, antero-dorsally in the region where the neuropore has closed, and about the same time two lateral ectodermal involutions form (Fig. 26, A, _at_), which become the atrial or peribranchial pouches, at first distinct, afterwards united in the mid-dorsal line to form the adult cloaca and atrial aperture. Ingrowths from the atrial pouches and outgrowths from the wall of the pharynx coalesce to form the proto-stigmata (primary gill-slits) by which the cavity of the branchial sac is first placed in communication with the exterior through the atrial apertures. Opinions differ as to whether only one or a few pairs of true gill-clefts are represented in the young Ascidian; and the actual details of their formation and subdivision, to form the stigmata of the adult, differ considerably in different forms. In _Clavelina_ the stigmata are formed as independent perforations of the pharyngeal wall; in _Ascidia_ two pairs of protostigmata increase to six pairs, which are subdivided into stigmata; _Botryllus_ and other forms are intermediate in some respects. No doubt the subdivision of proto-stigmata is primitive, but has been lost from the ontogeny in some cases. To what precise extent the walls of the atrial or peribranchial cavities are formed of ectoderm, or of endoderm, is still doubtful.

The embryo is hatched about two or three days after fertilisation, as a larva or Ascidian tadpole (Fig. 26, A) which leads a free-swimming existence for a short time, during which it develops its nervous system and cerebral sense-organs, and the powerful mesoblastic muscle-bands lying at the sides of the notochord (now a cylindrical rod of gelatinous nature surrounded by the remains of the original cells) in the tail which form the locomotory apparatus. Fig. 26, A, shows this stage, the highest in its chordate organisation, when the larva swims actively through the sea by vibrating its long tail with the dorsal and ventral fins.

In addition to the structures already mentioned, the mesoderm {60}has formed the beginning of the muscular body-wall, the connective tissue around the organs, and the blood; the endostyle has developed as a thick-walled groove along the ventral edge of the pharynx, which has become the branchial sac; and the pericardial sac and its invagination the heart have formed in the mesoblast between the endostyle and stomach. The "epicardiac tubes" grow out from the posterior end of the endostyle to join the pericardium. They play an important part in the formation of buds in the colonial Tunicata. The heart acquires a connexion with blastocoelic blood-spaces at its two ends. The heart and pericardium show the same relations in Tunicata as in Enteropneusta, but it is very doubtful whether these organs are genetically related to the Vertebrate heart.

The unpaired optic organ in the cerebral vesicle when fully formed has a retina, pigment layer, lens and cornea; while the ventral median organ is a large, spherical, partially-pigmented otolith attached by delicate hair-like processes to the summit of a hollow "crista acustica" (Fig. 26, A). After a few hours, or at most a day or so, the larva attaches itself {61}by one or more of the three anterior ectodermal glandular papillae (one dorsal and two lateral) to some foreign body, and commences the retrogressive metamorphosis which leads to the adult state. The adhering papillae, having performed their function, begin to atrophy, and their place is taken by the rapidly increasing test. The tail which at first vibrates rapidly is partly withdrawn from the test and absorbed, and partly cast off in shreds (Fig. 26, B, C, D). The notochord, nerve-tube, muscles, etc., are withdrawn into the body, where they break down and are absorbed by phagocytes. The posterior part of the nerve cord and its anterior end with the large sense-organs disappear, and the middle part or trunk-ganglion is reduced to form the relatively small ganglion of the adult, underneath which the hypophysial tube gives rise to the neural gland. While the locomotory, nervous and sensory organs are thus disappearing, or being reduced, the alimentary canal and reproductive viscera are growing largely. The branchial sac enlarges, its walls become penetrated by blood-channels, and grow out to form bars and papillae, and the number of openings greatly increases by the primary gill-slits being broken up into the transverse rows of stigmata. The stomach and intestine, which developed as an outgrowth from the back of the branchial sac at the right side, become longer and curve, so that the end of the intestine acquires an opening into at first the left hand side, and eventually the cloacal or median part of the atrial cavity. The adhering papillae have now disappeared, and are replaced functionally by a growth of the test over neighbouring objects; and at the same time the region of the body between the point of fixation and the mouth (branchial aperture) increases rapidly in extent, so as to cause the body of the Ascidian to rotate through about 180°, and thus the branchial siphon is carried to the opposite end from the area of attachment (see Fig. 26, B, C, D, E). Finally the gonads and their ducts form in the mesoderm between the stomach and intestine. We thus reach the sedentary degenerate fixed adult Ascidian with little or no trace of the Chordate characteristics so marked in the earlier larval stage (see E and A, Fig. 26). The free-swimming tailed larva shows the Ascidian at the highest level of its organisation, and is the stage that indicates the genetic relationship of the Tunicata with the Vertebrata.

In some Ascidians with more food-yolk in the egg, or in which {62}the development takes place within the body of the parent, the life-history as given above is more or less modified and abbreviated, and in some few forms the tailed larval stage is missing. Some exceptional cases of development will be noted below under the groups to which they belong.

The remarkable life-history of the typical Ascidian, of which the outlines are given above, is of importance from two points of view:—

1. It is an excellent example of degeneration. The free-swimming larva is a more highly developed animal than the adult Ascidian. The larva is, as we have seen, comparable with a larval fish or a young tadpole, and is thus a Chordate animal showing evident relationship to the Vertebrata; while the adult is in its structure non-Chordate, and is _on a level_ with some of the worms, or with the lower Mollusca, in its organisation, although of an entirely different type.

2. It shows us the true position of the Ascidians (Tunicata) in the animal series. If we knew only the adult forms we might regard them as being an aberrant group of Worms, or possibly as occupying a position between worms and the lower Mollusca, or we might place them as an independent group; but we should certainly have to class them as Invertebrate animals. But when we know the whole life-history, and consider it in the light of "recapitulation" and "evolutionary" views we recognise that the Ascidians are evidently related to the Vertebrata, and were at one time free-swimming Chordata occupying a position somewhere below the lowest Fishes.

{63}CHAPTER III

TUNICATA (_CONTINUED_)

CLASSIFICATION: LARVACEA—APPENDICULARIANS—STRUCTURE, ETC.—ASCIDIACEA—SIMPLE ASCIDIANS—SPECIFIC CHARACTERS—COMPOUND ASCIDIANS—GEMMATION—MEROSOMATA— HOLOSOMATA—PYROSOMATIDAE—THALIACEA—DOLIOLIDAE—SALPIDAE—GENERAL CONCLUSIONS—PHYLOGENY.

We now turn to the systematic classification of the group; and further details of structure or function, points of interest in the life-history such as budding and the formation of colonies, the habits and occurrence, and other peculiarities such as phosphorescence, will all be noted under the orders, sub-orders, families and genera in which they occur.

CLASS TUNICATA.

The Tunicata or Urochordata are hermaphrodite marine Chordate animals, which show in their development the essential Vertebrate characters, but in which the notochord is restricted to the posterior part of the body, and is in most cases present only during the free-swimming larval stages. The adult animals are usually sessile and degenerate, and may be either solitary or colonial, fixed or free. The nervous system is, in the larva, of the elongated, tubular, dorsal, Vertebrate type, but in most cases it degenerates in the adult to form a small ganglion placed above the pharynx. The body is completely covered with a thick cuticular test ("tunic") which contains a substance similar to cellulose. The alimentary canal has a greatly enlarged respiratory pharynx or branchial sac, which is perforated by two or many more or less modified gill-slits opening into a peribranchial or atrial cavity, which communicates with the exterior by a single dorsal exhalent aperture (rarely {64}two ventral apertures). The ventral heart is simple and tubular, and periodically reverses the direction of the blood-current.

This Class is divided into three Orders:—The Appendicularians, the Ascidians, and the Salpians (see Fig. 27).

ORDER I. LARVACEA (APPENDICULARIANS).

Free-swimming pelagic forms, in which the posterior part of the body takes the form of a large locomotory appendage, the {65}"tail," in which there is a skeletal axis, the urochord. A relatively large cuticular test, the "house," may be formed with great rapidity (in an hour or so) as a secretion from a part of the ectoderm; it is, however, merely a temporary structure which is soon cast off and replaced by another. The branchial sac is simply an enlarged pharynx with two ventral ciliated openings (stigmata) leading to the exterior. These may be regarded as the representatives of the primary gill-slits (undivided) of the Ascidian. There are thus a single pair. There is no separate peribranchial, atrial, or cloacal cavity. The nervous system consists of a large dorsally placed ganglion and a long nerve-cord, which stretches backwards over the alimentary canal to reach the tail, along which it runs on the left side (morphological dorsal edge) of the urochord. The anus opens ventrally on the surface of the body, usually in front of the stigmata. No reproduction by gemmation or metamorphosis is known in the life-history.