Tunicates or Sea Squirts: A Wet Link by Ronald L. Shimek, Ph.D.

In the last decade one of the most exciting, and active, areas of invertebrate zoological research has been the study of phylogeny, or the science of animal groups' evolutionary relationships. Biologists have always postulated about which animal groups were related and it has now become possible to actually determine those relationships. Within some groups such as the vertebrates (animals with backbones), this study has always been made a bit easier by a good fossil record which often can reveal useful intermediate groups. One of the best examples, the first fossil of Archaeopteryx, a classical un-missing "missing-link," was found in the mid-nineteenth century. This small animal was a bit bigger than a pigeon and, depending upon the interpretation of the moment, was either a small dinosaur with feathers, or a small bird with teeth, a long tail, and fingers. Dozens of other exquisite fossils, many of them recently discovered and described from southern China, have shown that many dinosaur lineages had feathers. Some of these lineages obviously lead to birds, while others had different descendents; some of the earlier progenitors of Tyrannosaurus rex, for example, had feathers. While the fossils of T. rex have come from sediments that are too coarse-grained to show either skin impressions or feathers on this delightfully cuddly animal, it is perhaps not too far fetched to hypothesize that adult T. rex may have retained some feathers as a signaling device, perhaps to signal dominance or sexual prowess. Imagine the remake of Jurassic Park showing a T. rex with a large, erective feathered crest, as a sort of Cockatoo from Hell. The taxonomic revision of the classification of the vertebrates resulting from these and other fossils is well underway, and the realization that dinosaurs and birds are simply different expressions of the same lineage is becoming commonplace, at least in the biological sciences.

Within the last couple of decades, researchers have made similar strides in recognizing the relationships within groups whose fossil record is sparse, primarily by comparing the genetic material found in several lineages. For such determinations, many different types of genetic material are examined and compared. Only within the last several years have sufficient data become available, together with the sophisticated statistical analyses necessary to compare them. The relationships resulting from such studies are helping to rewrite our understanding of the evolutionary history of life.

Biologists have always been able to examine living organisms by assessing characteristics, and have always been able to infer degrees of "relatedness." The problem with such assessments is that each biologist could assess things differently- and did. This process led to the development of several hypothesized evolutionary trees in the animal kingdom, with little actual hard proof for most of the branches on any of them. Often, these proposed evolutionary pathways were based on a great degree of logic. Unfortunately, few of these pathways were congruent, indicating that the application of a logical methodology was rather like the application of the concept of beauty. Logic, it seemed in this regard, was in the eye of the beholder.

New, less subjective methods of comparing animal groups started to become available in the mid-twentieth century, primarily in response to the problems resulting from previous subjective evaluations. These techniques were modified, becoming more sophisticated and quite methodologically rigorous. By the late 1980s, it was possible to evaluate many different character sets simultaneously and unambiguously, and from these evaluations, and by asking the appropriate questions, to infer a series of relationships between animal groups.

Within the last few years, then, several different and independent methods have simultaneously been used to evaluate the evolutionary history of the animal kingdom. By independence, in this regard, I mean unrelated ways of using different original data. These methods were:

These techniques have allowed the determination of relationships to a greater level of accuracy than heretofore. One of the more interesting outcomes is that all three methodologies effectively resulted in the same set of relationships, and some of these relationships were unexpected (Halanych, 2004). This has resulted in what appears to be a paradigm shift, similar to what took place in geology in the late 1960s when plate tectonics supplanted earlier concepts of geological activity.

One of the more interesting side-effects of these studies has become the more-or-less formal realization that the taxonomic hierarchy that has been in use for 250 years is seriously flawed, and in need of replacement. This has been apparent for some time, but the ball is finally rolling for its revision. In one of the most recent texts (Ruppert, et al., 2003), the terms of this hierarchy, such as phylum, class and subclass, are used only as informal descriptive terms. The problem is that if one is dealing with a group of interrelated organisms, animals in this case, where many of the connections between them are now known with some certainty, deciding how to group them becomes difficult. Basically, it is a question of "Where does one draw the line?" Some of the groups considered by many authorities to have phylum status as recently as five years ago are now considered to be part of some other group. Other groups, such as "The Phylum Platyhelminthes," or flatworms, have been shown to be a mistaken assemblage cobbled together from animals belonging to several unrelated lineages. While the term "flatworm" remains as a good descriptive term, the term Platyhelminthes no longer applies.

The animal kingdom now appears to be comprised of three major lineages and several smaller ones. Such lineages are the, sometimes, diverse descendents of a common ancestral organism and at what point the line is drawn dividing them into separate entities is the question of the moment. The most diverse of these lineages is called the Lophotrochozoa and includes all of the groups sharing a common larval type called a trochophore, a feeding apparatus consisting of an array of ciliated tentacles. The Lophotrochozoa consists of annelids, mollusks, and many of the smaller animal groups. The largest lineage, in terms of the number of species contained in it, is called the Ecdysozoa. These are animals that must molt to grow and whose name stems from ecdysis, the process of molting. These are the arthropods, nematodes, and some smaller groups. The final group is a lineage that is referred to as the Deuterostomia. This lineage consists of the echinoderms (sea stars, sea urchins and their kin), chordates (fish, mammals, amphibians, and their kin) and a few smaller groups.

The animal groups within the Deuterostomia also formed a group recognizable in earlier, more subjective schemes of relationships. In fact, the name was first proposed in the 1880s; however, the recent re-evaluation of animal groupings has lead to some significant shifts within it. Previously, biologists considered that the Deuterostomes formed a more-or-less simple linear evolutionary relationship. The echinoderms were considered to be the basal and ancestral group, giving rise to the hemichordates (acorn worms and some other small bizarre polyp-like animals), which, in turn, gave rise to the chordates, with the tunicates being some sort of ancestral invertebrate chordate. It now appears the basal group was some common unknown ancestor with intermediate characters. From this ancestor came two lines of descent, one lineage leading to hemichordates and echinoderms, and the other to chordates, with the tunicates as a separate offshoot of that branch. Interestingly enough, small fossils (and a reconstruction) assignable to the chordates have recently been found in some of the earliest Paleozoic rocks, well before the time that echinoderms became common or abundant. This would be expected if the echinoderms were a sister group of the chordates rather than an antecedent one.

Figure 1. The Deuterostome Animal Groups. Top: An older conception of the relationships within the deuterostomes. Bottom: A current conception of the relationships with the deuterostomes. Previous relations left out the odd wormy critter known as Xenoturbella, as so little was known about it. Recent genetic studies place it firmly within the deuterostomes. Modified from Halanych, 2004.

We used to think of the tunicates as a rather funky type of chordate. The major reason for this is their specific larva that looks a lot like a small, simplified fish. On this basis they were considered to be pretty closely related to actual fishes. Apparently, this is not the case. They now appear to be considerably more distantly related. While in one sense they are "some sort of halfway house" to chordates, they probably would be better considered as a successful lineage all their own, derived from a stock that gave rise, in time, to actual chordates, but not through the tunicates.

The group called the Tunicata contains some of the oddest invertebrates. All tunicates are marine animals and while none is found in freshwater, a relatively large number of species may live in estuaries. There are three subgroups of tunicates possessing some common characteristics. As do many other invertebrate groups, they possess a larva that undergoes a dramatic metamorphosis into a juvenile. As mentioned above, their larvae look like small tadpoles or very simplified small fishes, and possess in their tail a rod-like structure called a notochord that acts, in part, as an antagonist for their tail's musculature. Having such a rod in the tail allows them to swim much more efficiently than they could without it. The larvae and the swimming ability are transitory. Only in one class, the Larvacea, do the adults retain the tadpole shape, and even so, as adults they do not swim much. In the other classes, the larval tadpole shape, the tail, and its notochord are found only in the larvae. The notochord is one of the primary defining characteristics of the animal group called the Chordata; in fact, that structure gives the phylum which contains all the vertebrates, as well as some other animals, its name.

Figure 2. A diagram of a typical ascidiacean tadpole. Although the gut,
branchial basket, and gill slits are present, they are not functional and do not
open in the larva.

Showing other similarities to the chordates, all tunicates possess openings in the front part of their gut that are considered to be homologous with the gill slits of simple vertebrates such as lampreys. Additionally, most adult tunicates possess a rudimentary nervous system; as with many of their structures, however, the larval nervous system is more sophisticated, consisting of a main dorsal hollow nerve cord located above the notochord with very small nerves leaving it. This is a pattern also seen in the chordates. As their name implies, many possess a "tunic" or outer supportive/protective layer. This is comprised of a type of cellulose called tunicin (Millar, 1971; Kozloff, 1990; Ruppert, Fox and Barnes, 2003). Curiously, one of the few other places where cellulose is found in animals is as fibers deposited within the skin.

The three subgroupings may be referred to as the taxonomic classes of the Phylum Tunicata. Of these, only the Class Ascidiacea, commonly called sea squirts or tunicates, is likely to be found in aquaria. The other two classes, the Classes Larvacea (or Appendicularia) and the Thaliacea, although almost unbelievably common in the world's oceans, are wholly pelagic and exceptionally difficult to maintain for any but the shortest time in aquaria.

There are about 1200 species of ascidiaceans. The adults are all sessile, benthic dwelling, ciliary-mucous suspension-feeding animals. Although there are a great many completely solitary species, many others are gregarious, forming aggregations. These aggregations may form from "communal settlement" or as a result of "budding" of discrete new individuals. Other colonies may form when many zooids share a common tunic. All tunicates live fastened to the substrate by their cellulose tunic.

The tunic is wholly exterior to the animal, almost completely surrounding the body, and is secreted by the mantle, a specialized type of secretory epidermis. The tunic is composed of numerous chemicals, but includes tunicin, which is a type of cellulose, as mentioned above. Only the two siphonal openings, an incurrent and an excurrent for water passage into and out of the branchial basket, pierce the tunic. Unlike other animals with a surrounding cuticle, tunicates grow inside the tunic without molting. This is probably due to their ability to resorb and redeposit tunic materials at the mantle-tunic interface. Some species have channels in the tunic that are continuous with internal blood spaces, and allow for the secretion and redeposition of tunic material as well as for the secretion of defensive materials into the tunic.

The two tunic openings are the incurrent, or branchial, and excurrent, or atrial, siphons. Water enters the animal through the incurrent siphon and passes into the highly modified throat region called "the pharynx." The gill slits have become highly modified and numerous, turning the pharynx into a biological sieve called the branchial basket. The water flows through the gill slits into another internal chamber called the atrium. From the atrium, water exits the animal through the excurrent or atrial siphon. The atrium also contains the anal and reproductive openings. Gonadal products and feces are both blown out of the atrium through the excurrent siphon by the water currents generated by the branchial basket's cilia.

Cilia lining the edges of the gill slits pump water through the branchial basket. In this structure, mucus is produced from a ventral groove or gutter called the endostyle. A primary component of this mucus is indistinguishable from vertebrate thyroid hormone, and the endostyle is often regarded as the thyroid gland's evolutionary precursor. The mucus flows as a sheet from the ventral groove dorsally over the branchial basket's inner surfaces. If you have seen living tunicates, the terms dorsal and ventral may seem rather odd here, and are used only to denote "morphological" directions. The part of the branchial basket closest to the atrial siphon is considered dorsal, and the ventral edge is on the other side of it. The terms dorsal and ventral come from the orientation of the larvae, and really have little meaning in the adult due to the drastic metamorphosis that occurs between the larval and adult stages.

Very fine particulate organic material, mostly bacterioplankton or small phytoplankton, is filtered from the water and is caught on the mucous layer lining the branchial basket. Tunicates are awesomely effective suspension-feeders; even small ones can filter hundreds of liters of water per day and remove well over 95% of its bacteria.

Figure 8. A shallow water tunicate-dominated area off the British Columbia
coast. The small orange tunicates are the gregarious species, Metandrocarpa dura.
The elongate stalked forms are the solitary species Styela montereyensis. The smooth
gray forms, mostly in the background, are compound forms called Distaplia smithi.

Figure 9. Left: A specimen of a tunicate, Styela montereyensis, (see the preceding figure) dissected by one of my students several years ago. The cut and opened tunic is the white structure in the background. The branchial basket has been torn, but some of the structure is still visible. The gonads are found throughout the animal. Right: A portion of the branchial basket of this animal photographed through a microscope. The oval structures crossed by the horizontal bars are the filtering gill slits.

The mucus, with its adherent food, is moved by ciliary action to a food groove located on the dorsal midline of the branchial basket. From there it is moved, in the food groove, into the mouth. A very short esophagus transfers the food-laden mucus to a bag-like stomach. Associated with the stomach is a large structure called the digestive gland. The function of the digestive gland is not fully understood, but it probably contributes to digestion and absorption of food. The short, straight intestine leaves the stomach and leads to a rectum and anus. Feces are deposited in the atrium, and flushed out with the excurrent water passing out the excurrent siphon.

Several species of tropical colonial sea squirts contain symbiotic cyanobacteria growing in the branchial basket. These cyanobacteria, in the genus Prochloron, appear to function much like the zooxanthellae found in many other invertebrates.

The tunicates possess an open circulatory system; one that is largely without vessels. Blood flows through large tissue spaces or blood channels in the tissues. Arteries, veins, and capillaries are usually absent. They have a heart, but it is a simple tube with walls that contract to force the blood through it. Unlike the heart in most animals, this heart is capable of reversing its beat. Generally, the heart beats about a hundred times in one direction, stops for a moment and then beats about a hundred times in the other direction. Most tunicates appear to have channels for blood flow through the gills; these have sometimes been called blood vessels, but they have a very simple structure. Consequently, the gill region of the branchial basket probably functions as a respiratory organ, but little direct evidence supports this.

In some squirts, the blood contains odd rare-earth chemicals, commonly vanadium or niobium. These metals were once thought to assist in respiration, but are now known to be anti-fouling or anti-predator defenses (Stoecker, 1980; Young, 1986). Other chemicals that appear to be primarily defensive are found in the blood. Blood serum containing these metals and other noxious chemicals either leaks through the epidermis, or is secreted by it and oozes through the tunic. Additional, exceedingly acidic, defensive chemicals are found in the tunic secretions of a few species. These secretions have pH values of about one.

Many of the organs or organ systems present in many animals are reduced or absent in adult tunicates. They have no specialized excretory or osmoregulatory organs. They deposit their nitrogenous wastes as solid crystals of guanine or uric acid in the tunic. This is termed "storage excretion," and the animals often look like they are dusted with a fine layer of white dots, resulting from these granules in the tunic. The reduction of systems is also reflected in the nervous system, which is reduced to a small ganglion generally located between the siphons, above the atrium. Additionally, most adult tunicates have little capability for movement or locomotion; however, colonies in several tropical genera including Didemnum are capable of regular movement. These move by secreting new tunic in the direction of locomotion and releasing old tunic at the posterior end. The tropical genera that are mobile all contain cyanobacteria (Birkeland, et al., 1981) which may provide them some additional energy to use for motion.

Some temperate compound ascidiaceans, such as Eudiastoma or Diplosoma species, also move by colony expansion. The colony grows rapidly and may send out large blob-like processes over the bottom. Connections between these processes and other parts of the colony may break and the smaller fragments may then grow new extensions, and the process is repeated. While any single part of the colony remains in place, the colony moves by extensions that move out over the substrate.

All tunicates are hermaphroditic, and are often self-fertile. The gonads can develop just about anywhere in the animal. The gonoducts run parallel to the intestine and empty into the atrium near the anus. Gametes released from the gonoducts develop into non-feeding "tadpole" larvae.

These larvae look very much like tiny tadpoles. They have a small, globular body with a long tail. The tail contains a supportive gelatinous rod, the notochord, composed of large cells. There are muscles on either side of the tail and the notochord. The notochord acts as an internal hydrostatic skeleton for the antagonism of muscle flexion. These small larvae swim rather well. A fin is typically found running completely around the length of the tail along its midline. The dorsal, hollow nerve cord runs above the notochord from a hollow, anterior brain to the tip of the tail. There are typically a single eye, or photoreceptor, and a statocyst, or balance organ, associated with the brain. The gut is present but as the larva is non-feeding both the mouth and the anus are sealed. Generally, these larvae have only a few, non-functional gill slits. Several adhesive papillae are found on the animal's front end.

The larvae exit the parent's atrial siphon and swim for a short period before selecting a substrate, often by making exceptionally precise choices (Svane and Young, 1989; Young, 1989; Stoner, 1994). During this period they are often conspicuous and might appear to be prime food for planktivorous fishes. This does not appear to be the case, however, and it appears that some of the larvae are also protected by toxic chemicals (Lindquist, et al., 1992; Lindquist and Hay, 1996). The larvae glue themselves, nose first, to the substrate using the secretions of anterior adhesive papillae. This is followed by an exceptionally rapid, drastic and complex metamorphosis. Epidermal cells covering the animal's surface contract and within a period of 10 to 15 seconds they crush the tail and its inner notochord, muscles, brain, nerve cord, and sensory structures into a mass of tissue debris. The rest of the viscera rotate about 90 degrees by differential growth, and the mouth and anus open. The dorsal ganglion develops from nervous system remnants. Little juvenile squirts start to feed a few days after settlement. Growth can be very rapid in small animals, with adult size reached in a few weeks. Others live longer and grow more slowly (Cloney, 1982).

The largest solitary animals, reaching sizes in excess 30 cm in height, may live for several years. Most solitary species are much smaller, and are probably more ephemeral. Colonial tunicates grow by budding new individuals off the initial individual. There is almost no polymorphism, and the small individuals that make up the colony live inside a common tunic. These colonies can be huge; I have seen some in the North Pacific that have been 20 m long and 3 to 4 m wide, and they can grow very rapidly. On occasion, I have peeled back the edge of such a colony and found living sea cucumbers and scallops entombed under it.

Figure 12. Tunicate wars. The gray Diplosoma sp. is overgrowing
the orange Ritterella sp. Overgrowth kills the loser.

Tunicates are often strikingly beautiful animals, but they have been difficult to maintain in many aquaria for the same reason that many other suspension-feeding animals are. As with many of these filter feeders, tunicates are primarily bacteriovores, and in general, our systems simply don't contain bacteria or fine particulate material sufficient to support their growth.

If the conditions for normal growth are met, there are no reasons the animals should not reproduce in aquaria. Asexual reproduction of the colonial forms is common in nature and should occur in aquaria. Sexual reproduction may occur as well, particularly in the colonial forms. The short larval lives, often only a few minutes, of some of these animals will facilitate their reproduction in our systems. If conditions are acceptable for the larvae, they will settle and metamorphose into juveniles. The recently settled juveniles are very vulnerable to predation, and may not survive in a tank with many grazers or foraging hermit crabs. Once past the smaller juvenile stages, however, these animals are generally well protected from predation by their chemical and physical defenses (Stoecker, 1980; Young, 1986) and will survive well in aquaria.

Tunicates, like corals, will be kept for the aesthetic value of the colony or individual. They generally don't move, and as adults they have almost nothing that could be called behavior; about all they can do is open and close their siphons. Nonetheless, for the hobbyist who has a "green thumb," tunicates can provide much colorful beauty to a tank. In purchasing tunicates, it is important to choose small individuals, and generally individuals that are attached to rocks or some other substrate. In my experience, large individuals do not seem to survive the collection and transport process well, and additionally, when they feed they often need far more nutrition than is found in a normal tank. Although loose individuals can be fastened to rocks with epoxy or other glues, the collection process often damages them when they are removed from their substrate.

For more information on these animals, literally dozens of web pages are devoted to tunicates, mostly to photographs of them; run searches using: ascidian, tunicate, or urochordata. Additionally, a lot of good up-to-date technical information can be gathered from the issues of the Ascidian News.