The ability of ascidians to regenerate all or part of their body was noted over 100 years ago. Interest in this has grown of late because of the potential of pluripotent stem cells for treating various human maladies such as spinal cord injuries. Regeneration is likely related to asexual reproduction by budding. Even so, solitary ascidians that don’t bud, such as Ciona, can also regenerate parts of the body. This is particularly useful because they cannot escape predators. Thus, if a fish bites off part of a siphon or even the neural complex, they can regrow the missing parts. A particularly critical experiment was done in 1964 by G. Freeman, who showed that gamma irradiation inhibited cell division and budding in a colonial ascidian, and that injection of blood cells from an unirradiated individual of the same species into the irradiated one restored budding. These pluripotent blood cells can, therefore, give rise to all the body parts in the right position; just how they can do this is a hot topic for research.

Ascidian embryos undergo rapid development to a tadpole larva. Cleavage is determinant, meaning that cell fates become fixed very early in embryogenesis. If the first two cells are separated, each develops only into the cells that it would normally form in the intact egg. Cytoplasmic movements within the zygote shortly after fertilization set aside cytoplasm, known as myoplasm, that will form most of the larval tail muscle. In Ciona, hatching to a tailed, tadpole larva occurs within 24 hours, depending on the temperature (the total time from embryo to fertile adult is just three months). The larva swims for a few days at most until encountering a suitable substratum to which it attaches by anterior adhesive papillae. The tail is resorbed, and within about two days of settlement, the larva completes metamorphosis into a juvenile, with incurrent and excurrent siphons and two gill slits. Ascidians typically reproduce sexually at regular intervals during their life span, which can be a year or more. In colonial species, embryos are often brooded.

The size range of ascidian zooids is very wide. The solitary adults of phlebobranchs and stolidobranchs usually are fairly large, up to 6–7 cm in height, while the carnivorous ones can be up to 26 cm in height. In contrast, aplousobranchs, which, except for the solitary Ciona spp. ( Figures 1 and 2 A), are colonial and usually have very small zooids, just a few millimeters high. In spite of this enormous variation in size, reproduction and mode of feeding, the basic body plan is relatively conserved. The typical body plan of a zooid is shown in Figures 1 and 2 A. Feeding occurs as water enters via an oral or buccal siphon, passes through a branchial basket perforated by numerous gill slits, and exits by the excurrent siphon. The endostyle within the branchial basket secretes mucous, which traps the particles and carries them into the stomach. The nervous system is reduced to a dorsal ganglion, but there are numerous ectodermal sensory cells. The heart is located basally near the stomach, and periodically reverses direction. The blood flows from the heart through hemocoel spaces. All ascidians are hermaphroditic; self-fertilization is avoided in at least some simultaneous hermaphrodites, such as Ciona, by incompatibility of genetic variants of cell-surface proteins.

While tunicates are almost all filter feeders, eating phytoplankton and other small particles, there are several deep-sea species of ascidians, chiefly in the family Octacnemidae (Phlebobranchia), in which the oral or incurrent siphon is enlarged to form a mouth that can capture large prey. Because adult ascidians are fixed to the substratum, they invite predation by fish and other carnivores. As a result, they have a variety of chemical defenses, most thought to be synthesized by symbiotic bacteria, which presumably act to deter predators, and some ascidians concentrate vanadium, which has been shown to deter predatory fish.

(A) A solitary ascidian. Coloniality has evolved several times in ascidians. Adults are sessile. Arrows indicate water flow into the incurrent and out of the excurrent siphon. They are simultaneous hermaphrodites, but self-sterile due to gamete surface protein incompatibility. The larva undergoes a short pelagic phase before attaching to the substratum by the adhesive papillae; the tail is then resorbed and the nervous system remodeled. (B) Appendicularians, most only a few millimeters long, are exclusively pelagic. They secrete a series of houses for buoyancy and filter feeding. None is colonial. All except Oikopleura dioica are hermaphroditic. Larval development is direct. Tunicates have hearts that periodically reverse the direction of the blood flow. (C–E) The three groups of thaliaceans. (C) Pyrosomes form colonies up to several meters long, which move slowly by jet propulsion. Each zooid in the colony reproduces asexually, enlarging the colony, and sexually, producing an embryo termed a cyathozooid brooded within the colony. Each cyathozooid is resorbed after budding four zooids; this colony is expelled from the parent colony to start a new one. (D) Salps have a biphasic life cycle; the solitary zooid repeatedly buds off chains of zooids, called aggregates, each of which typically has just one egg, fertilized as soon as the zooid expands and begins pumping water. The eggs develop directly into small solitary zooids and are then expelled. Sperm are produced about the time the embryo matures. (E) Doliolids have a very complex life cycle. The larva develops into a non-feeding oozoid or nurse, which then produces a stolon that bears two types of zooids, trophozooids, which only feed and stay attached to the stolon, and phorozooids, which break off the stolon. The phorozooids then bud off gonozooids, each of which develops an ovary and a testis. They are distinguished from salps by muscle bands that are continuous around the body. (A) Adult image ©BIODIDAC; both adapted by Jon Houseman, Wikimedia Commons. (B) Adult image adapted with permission from SAHFOS. (C) Colony and Adult zooid images adapted with permission from Anne and Wilfried Bay-Nouailhat; cyathozooids after Godeaux, J. (1987); (D) after Kim et al. (2012); (E) adapted from Braconnot (1971).

Although all adult ascidians are sessile and most have a tadpole larva with the characteristic chordate features of a notochord and a dorsal, hollow nerve cord, a number of features have evolved repeatedly among all three ascidian suborders, the Stolidobranchia, Aplousobranchia, and Phlebobranchiata ( Figures 1 and 2 A ). For example, tailless larvae have evolved at least five times within the stolidobranchs. Furthermore, coloniality resulting from asexual reproduction by budding of the original sexually-produced zooid has evolved independently several times. In some instances, the asexually produced zooids remain closely adherent in a common tunic and in others, they stay attached only through narrow stolons. When colonies of the same, but not different, genotype of the styelid ascidian Botryllus expand to touch each other, they fuse; however, colonies of the aplousobranch ascidian Diplosoma listerianum fuse regardless of genotype.

In O. dioica, development to adult takes just 24 hours. Gametogenesis is rapid, with oocyte nuclei and polyploid nurse nuclei in a syncytium termed the coenocyst. At the end of oogenesis, the oocytes are individualized and begin to undergo the meiotic divisions. Fertilization occurs at first meiotic metaphase. There are cytoplasmic movements after fertilization, and it seems likely that they may also segregate the myoplasm, though this aspect of embryogenesis has not been studied in appendicularians. Gastrulation in O. dioica occurs one cell division earlier than in Ciona. At the end of development, the tadpole hatches and its tail rotates 90° and begins to beat and inflate the first house.

Not only do appendicularians have simple, tadpole-like bodies ( Figure 1 B), their reproductive strategy is also streamlined. They all reproduce exclusively sexually and, with the exception of Oikopleura dioica, they are hermaphroditic, producing eggs and sperm sequentially, thereby avoiding self-fertilization. Their life spans are very short: that of O. dioica, the only appendicularian that has been cultured in the laboratory, is 10 days or less, depending on temperature. When reproducing, they swim out of the house towards the water surface. In the wild, over a thousand individuals per liter have been documented. Fertilization is external, and after shedding the gametes, the adults die.

It is still a bit of a mystery how such small, simple animals can make such elaborate houses. The ectoderm cells that secrete the house, termed the ‘oikoplast’, are polyploid; the glycoproteins that these cells secrete, known as ‘oikosins’, are unique to appendicularians. Specific regions of the oikoplast secrete different oikosins, but how they form the different mesh sizes is a major unanswered question.

Appendicularians are in some ways the simplest tunicates. Most are very small, about 2 mm long, and as adults they resemble the tadpole larva of ascidians, with a trunk, motile tail, notochord and nerve cord; hence their alternative name, Larvacea ( Figures 1 and 2 B). But they have also evolved some extremely bizarre novel traits, including secretion of a ‘house’ around themselves which serves in filter feeding and provides buoyancy ( Figure 1 ). This house has meshes of varying sizes that concentrate particles and filter out ones too large to fit in the mouth. The beat of the tail draws water through the house and into the mouth. When the house becomes clogged with particles, the animal swims out of the house and inflates another, which has already been secreted and lies deflated around the trunk until needed. The structure of the house is very intricate, with meshes of different sizes. Moreover, the morphology of the house differs among the various genera. Particularly large appendicularians with trunks 0.5–1.0 cm long, such as Bathychordaeus and Mesochordaeus from Monterey Bay, California, have houses up to 30 cm in diameter.

Thaliaceans: salps, doliolids and pyrosomes

Although thaliaceans appear to be monophyletic, they are quite divergent from one another, as well as from ascidians and appendicularians. They have all lost the dorsal, hollow nerve cord and notochord, except for a rudimentary one in some doliolid larvae. Their reproductive strategies are particularly strange, with very complex alternation of asexual and sexual generations ( Figure 2 C–E). The selective advantage of asexual reproduction in thaliaceans is presumably that it allows them to exploit blooms of phytoplankton to rapidly expand the population. Because thaliaceans live in the open ocean, they are difficult to maintain in the laboratory and have been much less studied than ascidians or even O. dioica.

Salps are usually very scarce in the ocean, but when there are blooms of phytoplankton, the populations can explode. Net tows have documented over 1,000 individuals of Salpa thompsoni per 1,000 cubic meters in Antarctic waters. Together with appendicularians and doliolids, they are major players in the marine food web, cycling energy from shallow waters deep into the water column by eating phytoplankton and excreting carbon-rich fecal pellets, which together with their dead carcasses sink down into the water. The carcasses are colonized by bacteria, which in turn are colonized by viruses. Together with the discarded houses of appendicularians, the bodies of dead pelagic tunicates make up a substantial proportion of the so-called ‘marine snow’. Salps are parasitized by the amphipod Phronema, which eats the insides of the zooids and uses the tunic as a house as it moves through the ocean.

Salps can be quite large: while individuals of a nearshore species, Thalia democratica, are only 1–2 cm in length, those other species range up to about 10 cm long. They typically undergo a diurnal migration, going as deep as several hundred meters during the day and coming up to the surface at night, where reproduction, both sexual and asexual, occurs.

Salps move through the water by rhythmic contractions of muscles that nearly (but not quite) encircle the zooid and pump water in through the mouth and out the cloaca. The individual zooid produced by union of egg and sperm is termed the solitary form ( Figures 1 and 2 D); it periodically buds off a chain of up to 200 asexually generated individuals, termed aggregates, each of which contains an oocyte (egg) and a testis. As the chain is extruded, the individuals at the tip of the chain inflate and begin to pump water. Inflation of zooids progresses from the tip to the base of the chain. Each aggregate typically produces a single egg (rarely two), which is fertilized internally as soon as the zooid begins to pump; the embryo is brooded. Thus, a given chain has zooids with embryos at successive stages of development.

Typically, after release from a solitary zooid, the chain breaks up into individuals. The embryos grow into mature solitaries and swim out of the cloaca of the parent. Then the testis of the aggregate sheds sperm, after which the zooid dies. The sperm, which are drawn into the branchial chamber of the newly inflated aggregates, find their way to where the ‘oviduct’, a column of cells with no lumen, meets the branchial chamber. Oddly, the corkscrew-shaped sperm burrow through the centers of the cells of the oviduct to find the egg. Early development in the ovary is quite bizarre. After the third division of the embryo, the blastomeres separate and non-germinal follicle cells termed calymnocytes move in between them. Development must, therefore, be determinant. The calymnocytes apparently function to nourish the blastomeres, which eventually come back together to form an embryo. Salps can live for months to a year or more.

Doliolids are also major players in the ocean food web, though they are generally smaller than salps, being rarely longer than 4–5 cm. They are preyed upon by copepods. High densities have been documented, for example, up to 550 individuals per cubic meter of Doliolum dentaculatum in the Yellow Sea. Doliolids are distinguished from salps by having muscles that entirely encircle the body. Instead of pumping water through the body to filter feed and move through the water like salps, doliolids glide through the water propelled by the action of cilia on the branchial basket. At intervals, their muscles contract and they jerk ahead.

Doliolids have an incredibly complex life cycle, the most complex of any tunicate ( Figure 2 E). The sexually produced individual, called the nurse, deriving from union of egg and sperm, does not directly feed, but produces a stolon which bears three rows of zooids ( Figures 1 and 2 D). Those in the center are called phorozooids, while those on either side are trophozooids or gastrozooids. The latter remain attached to the stolon and filter feed, nourishing the nurse and the growing phorozooids. The phorozooids detach from the stolon and bud off gonozooids, which are hermaphroditic. The gonozooids become sexually mature and typically release a few eggs, which are fertilized either within the branchial cavity of the doliolid or after being shed into the sea water. Then the zooids release sperm. Fertilization has not been observed. At 20°C, the entire life cycle can take about 20 days. Development is semi-direct. Doliolum embryos develop a tail, in some respects like that in ascidians, which has a notochord flanked by muscle cells but lacks a nervous system. The tailed larva does not hatch, and it is likely that tail movements, if any, are rather weak. The anterior portion of the embryo develops directly into a young adult, and the tail resorbs.

Pyrosomes are never common. Although the individual zooids are very small, like those in colonial ascidians, the colonies, consisting of thousands of asexually-produced zooids, can become very large, up to about 20 m long ( Figures 1 and 2 C). The colony is closed at one end, and open at the other; the opening can be up to nearly 2 m in diameter. A dead penguin trapped inside a pyrosome colony has been reported, and there are photos of divers with their heads inside pyrosomes.

Each small pyrosome zooid is oriented with the mouth pointing to the outside of the colony and the cloaca pointing inward. Water moves into the mouth and out through the cloaca and is expelled through the opening of the colony, driving it forward by slow jet-propulsion. Colonies of pyrosomes are most abundant in subtropical and tropical waters where they are often eaten by sea turtles. They are sometimes pink, and fishermen have called them ‘silk stockings’. Each zooid has light organs, which luminesce brilliantly when the colony is stimulated mechanically or by light. It is unclear whether this luminescence is due to symbiotic bacteria or not. A bacterial-type luciferase has been isolated from pyrosomes; bacteria have been seen in the light organ, but none have been cultured.

Pyrosomes have always been thought to be fairly closely related to ascidians, and recent phylogenetic analyses have placed them together with the other thaliaceans as sister group to the stolidobranch ascidians (Molgulidae, Pyuridae and Styledae). They are hermaphroditic, reproducing both sexually and asexually, with internal fertilization. The embryo cleaves into a group of cells termed the cyathozooid, which, before it is released into the sea water, buds off four zooids. Budding continues after release from the parent until the colony becomes quite large.