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Botrylloides (Milne Edwards, 1841)

Ilha Byrne 2020


The Ascidiacea, commonly referred to as 'sea squirts', are a class of sessile marine invertebrates within the subphylum Tunicata of the phylum Chordata (Brusca, Moore & Schuster, 2016). Ascidians form an important segment of the benthic food chain as they are a food source for many small invertebrates (Simkanin et al., 2013). The ascidian family Styelidae (styelids) is composed of the genera Botrylloides and Botryllus, which are commonly referred to jointly as botryllids. The genus Botrylloides is comprised solely of colonial ascidians which consist of tiny zooids embedded in a common tunic (Rupert et al., 2004) (Table 1). In members of this genus, zooids bear a buccal (oral/incurrent/inhalant) siphon but lack individual atrial (cloacal/excurrent/exhalant) siphons (Table 1). Instead, they possess an atrial opening that is characteristically large with a broad dorsal 'lip' extending from its anterior rim (Rocha et al., 2019). The zooids in a colony function as a single entity and remain attached to a variety of substrates from rocks and shells to man-made structures (Carver, Mallet & Vercaemer, 2006). Some species from the genus Botrylloides are pervasive global invaders (Table 1) and can be particularly damaging to the aquaculture industry (Carver, Mallet & Vercaemer, 2006). Botrylloides spp. are increasingly used as model organisms for studies in allorecognition, chimerism, senescence and angiogenesis (Blanchoud et al., 2018). 

The following webpage synthesises the most important information pertaining to this genus, with a particular focus on its Australian representatives. Several specimens were found on settlement plates deployed in Moreton Bay, Queensland in 2019-2020 (Figure 1). It should be noted that the taxonomic identification of ascidians is particularly challenging in the absence of anatomical and genetic analyses. Morphological characteristics were used to assign the specimens to the genus Botrylloides, so their identification cannot be conclusively resolved at this time. 

Table 1: A list of common terms used throughout this webpage. 




An individual that can exist as a single animal or as a part of a colony.

Colonial ascidian

A colonial organism consisting of several genetically identical individuals (zooids) that share a common vascular system.


Groups of two or more zooids arranged in certain configurations.


The development of a new zooid via asexual budding.


The first zooid derived from an ascidian tadpole larva.


Zooids created via asexual budding of the oozooid.

Primary bud

The bud directly attached to the adult zooid.

Secondary bud

The budlet linked to the primary bud.

Vascular system

The circulatory system that pumps blood and nutrients through the colony.  

Invasive species

A species that occurs outside of its natural range.


A structure through which water is drawn into or expelled out of the organism.

Figure 1

Physical Description

Tunicates are named after the polysaccharide-containing ‘tunic’ in which they are encased (Monniot, Monniot & Laboute, 1991). The individual zooids of a botryllid colony are arranged within this gelatinous matrix and tend to form flat sheets on a variety of substrates (Rinkevich et al., 1993). In members of the genus Botrylloides, the zooids are organised in meandering or ladder-like systems (refer to 'Classification' section), that share a common vascular system (Mukai, Saito & Watanabe, 1987) (Figure 2). The atrial opening of an individual zooid is wide and the upper section of the opening projects outward forming a ‘lip’ (Kott, 1985) (Figure 3). The zooids are positioned so that their atrial openings join to form shared excurrent siphons (Figure 4). Colonies are extremely variable in structure and appearance which is affected by several intrinsic factors. The size, maturity and arrangement of systems as well as the stage in the blastogenic cycle at which colonies are observed can influence appearance (Kott, 1985). Ascidians from this genus adopt several colour morphs and species are difficult to distinguish in the field. As an example of this challenge, Botrylloides violaceus (Oka, 1927) has been wrongly identified as B. leachii (Savigny, 1816), B. diegensis (Ritter & Forsyth, 1917) and B. aureus (Sars, 1851) on several occasions (Carver, Mallet & Vercaemer, 2006). 

Figure 2
Figure 3
Figure 4



Members of the genus Botrylloides are widely distributed and tolerant of a wide array of environmental conditions (Carver, Mallet & Vercaemer, 2006). However, they are most often found in sheltered areas and appear on both natural and artificial substrates (Carver, Mallet & Vercaemer, 2006). Colonies are rarely found in the intertidal zone as they are susceptible to desiccation (Rinkevich et al., 1993). Botrylloides spp. are distributed in coastal waters and are most prominent during the summer months in Australia (Atlas of living Australia, 2020; Westerman, 2003). 

Predation and defence

Colonial ascidians are consumed by crabs, shrimp, sea-stars, nudibranchs, small fish and flatworms (Simkanin et al., 2013). Ascidian larvae are most susceptible to predation as they are easily ingested by suspension feeders and planktonic predators (Simkanin et al., 2013). Species such as Botrylloides violaceus evade predation via chemical defences. Chemical constituents render the colonies unpalatable and consequently protects them against predation (Pisut & Pawlik 2002, Tarjuelo et al., 2002). Species that produce secondary metabolites are of particular interest to the pharmaceutical industry. Compounds originating from marine organisms, such as ascidians, have been used in drugs that combat cancer, viruses and bacteria (Shenkar & Swalla, 2011). 


Invasive species often exhibit useful traits such as resistance to air exposure, temperature and salinity variation (Rocha et al., 2019). Botrylloides spp. are extremely invasive as they are able to grow rapidly and exploit novel environments (Carver, Mallet & Vercaemer, 2006). Their ability to reproduce asexually and rapidly generate zooids through budding makes them efficient invaders. Budding can also result in the fragmentation of colonies and subsequently increase local abundance and dispersal potential (Carver, Mallet & Vercaemer, 2006). Colonial tunicates are also less susceptible to predation as they are mechanically and chemically defended, and are adept at overgrowing other organisms (Carver, Mallet and Vercaemer, 2006) (Figure 5). Botrylloides spp. have been known to outcompete other sessile invertebrates even under unfavourable conditions, such as high heavy metal and sewage concentrations (Carver, Mallet & Vercaemer, 2006). 

Invasions are facilitated by the dispersal of colony fragments or larvae on floating debris. Ships also act as vectors of dispersal as colonies often foul hulls and anchor chains and can be transported in the ballast water (Carver, Mallet and Vercaemer, 2006). Colonies establish on a wide range of substrates, including ropes, iron chains, seagrass and sponges (Rocha et al., 2019). Thick colonies often form on boats and piers, making cleaning particularly arduous and expensive. When they dominate substrates, they can overload floating equipment and smother native species (Rocha et al., 2019). They pose a particular threat to the aquaculture industry as they smother shellfish and cover sea cages (Carver, Mallet & Vercaemer, 2006). Some species, such as B. violaceus, are able to reattach themselves to substrates following disruption. This makes it difficult to mitigate invasions of this species using high-pressure washing, which is commonly adopted in the aquaculture industry (Bullard et al., 2007; Bock et al., 2011).
Figure 5


Ascidians are very effective filter feeders and can act as local biofilters (Hughes et al., 2005). They are capable of sequestering diatoms, phytoplankton, bacteria and other suspended particulate matter (Hughes et al., 2005). Most studies on filtration rates have focused on solitary ascidians and it would seem that they are more effective at significantly influencing water quality (Hughes et al., 2005). However, the filtration capacity of Botryllids certainly warrants investigation as other colonial ascidians have been used to remove harmful bacteria in aquaculture systems (Stabili et al., 2016).  


Botrylloides leachii employs one of the most specialised hibernation strategies exhibited by representatives of the phylum Chordata (Hyams, 2017). Colonies exposed to environmental stressors enter a state of torpor, in which zooids are absorbed and all feeding and reproduction ceases (Hyams, 2017) (Figure 6). The process appears to occur in seven stages: in the first two stages, blastogenesis occurs more rapidly and the zooids start to be absorbed. Hibernation occurs in stage three (Figure 6), whereby the colony is reduced to a carpet-like mass of vasculature. In stages four to seven the colony 'awakens' once favourable conditions arise. As the process progresses, proteins such as PIWI, PL-10 and PCNA are expressed more intensely in the cells of the endostylar region of the zooids and later in the remaining vasculature of the colony (Hyams, 2017). These last four stages are characterised by vascular budding and the development of new zooids, allowing the colony to regain full functionality (Hyams, 2017). The ability to hibernate provides B. leachii with a competitive advantage over other species and further justifies their success as an invader. 
Figure 6

Life History and Behaviour


Ascidians are mucus filter feeders that extract particulate matter from the water column. Seawater is drawn in through incurrent siphons and pushed through the ciliated pharyngeal gill slits (Millar, 1971) (Figure 7). The juncture between the pharynx and the buccal siphon is lined with buccal tentacles that prevent large food particles from entering the pharyngeal basket (Millar, 1971; Ruppert et al., 2004). The cilia on the stigmata (gill slits) create water currents that aid the movement of the food-laden water through the organism (Figures 8 & 9). Smaller food particles are captured by mucus lining the pharyngeal basket. The mucus is produced by the endostyle (Figure 7), which is located posterior to the pharyngeal basket (Brusca, Moore & Schuster, 2016). This mucus feeding net is very efficient and can trap particles as small as 0.4 µm (Carver, Mallet & Vercaemer, 2006). The food-bearing mucus is wound into a cord and moved across the internal surface of the branchial sac by ciliary movements into the stomach (Figures 8 & 9). Digestive enzymes originating from the gut wall aid the breakdown of food in the stomach. Water and waste products are eventually ejected through the atrial opening (Millar, 1971) (Figure 7). 
Figure 7
Figure 8
Figure 9

Life cycle

Members of the genus Botrylloides are hermaphrodites and can reproduce both sexually and asexually (Okuyama & Saito, 2001) (Figure 10). Sexual reproduction usually occurs in summer, while asexual budding is more pronounced in winter. In Botrylloides spp., asexual reproduction is not completely terminated during sexual reproduction as it is in other ascidian groups (Berrill, 1947). Once sexual reproduction results in the successful metamorphosis of a tadpole larva, colony growth via asexual palleal budding occurs (Berrill, 1947). Budding appears to be temperature-dependent in the species that have been studied extensively. However, once a critical minimum environmental temperature is reached, factors such as food availability, competition, salinity and turbidity are more important determinants of survival, growth and reproduction (Westerman, 2003). 

Figure 10

Sexual reproduction

Sexual reproduction occurs through cross-fertilisation with the eggs and sperm (see 'Reproductive system' section) produced asynchronously to prevent self-fertilisation (Milkman, 1967). In fact, there is evidence to suggest colonial tunicates can actively select sperm from fitter and more compatible conspecifics (Bishop et al., 1996). The fertilised eggs are brooded in a pouch (Figure 11) and extra-embryonic nutrients are supplied by the secretory lining of the pouch in some species, such as Botrylloides leachii (Mukai, Saito & Watanabe, 1987). 

This genus possesses great diversity in reproductive strategies with species such as Botrylloides lenis (Saito & Watanbe, 1985) and Botrylloides violaceus exhibiting extreme viviparity. In viviparous species, gestation is prolonged, and embryogenesis lasts longer than in ovoviviparous species (Mukai, Saito & Watanabe, 1987). Ovoviviparity coupled with short brooding periods seems to represents the primitive condition in this genus, with species progressing to viviparity (Saito & Watanabe, 1985). Under all circumstances, eggs undergo total cleavage (Monniot, Monniot & Laboute, 1991) and usually develop into tadpole larvae (Figure 12) within roughly 5 days (Mukai, Saito & Watanabe, 1987). The release of the larva from the brood pouch seems to be dictated by light levels, with most styelids, such as Botrylloides niger (Herdman, 1886), releasing their larvae in the morning (Svane & Young, 1989) (Figure 13). 

Compound ascidians often release fewer, more developed tadpole larvae than solitary ascidians (Svane & Young, 1989). The larval tail (Figure 12) consists of muscles arranged in longitudinal bands, allowing muscular flexion and thus movement through the water column (Svane & Young, 1989). Larvae exhibit a short planktonic period, with very few species experiencing a pelagic period lasting longer than a few hours (Berrill, 1947). The tadpole larvae of most ascidians possess a statocyst and an ocellus, capable of gravity and light reception, respectively (Berrill, 1947). However, in styelids, a compound 'photolith' is present that incorporates features of both aforementioned sensory structures (Svane & Young, 1989; Sorrentino et al., 2000). The photolith is particularly important during settlement as these larvae are negatively phototactic during the latter part of larval life (Kott, 1985).
Figure 11
Figure 12
Figure 13


Once the larva settles, three adhesive papillae are used to attach to the substrate and the larval tail regresses (Monniot, Monniot & Laboute, 1991). Larval structures are consequently destroyed and the body axis of the animal rotates 90º (Monniot, Monniot & Laboute, 1991) (Figure 14). The zooid organs arise from corresponding structures in the larva and the number of stigmata (gill slits) increase (Ruppert et al., 2004). Once the larval cuticle molts, the siphons can access the water column and the oozooid (juvenile) can commence feeding (Figure 14). Colony formation and growth can consequently proceed via asexual reproduction (Sabbadin, 1955; Millar, 1971).
Figure 14

Asexual reproduction

Asexual reproduction occurs primarily via palleal budding in Botrylloides spp., which given optimal conditions, can allow colony size to double weekly (Blanchoud et al., 2018). Buds (primary buds) can arise on either side of the atrial wall of each zooid and budlets (secondary buds) form via the same process on the primary buds (Figure 15). The development of buds in the same generation occurs simultaneously and in a coordinated manner across the colony (Berrill, 1947). The buds initially develop as disks of thickened atrial epithelium that are only three-four cells in diameter (Berrill, 1947). The buds grow to up to 12 cells in diameter and those that develop into sexually mature zooids are the largest (Berrill, 1947). As the bud grows, the inner vesicle starts to exhibit infoldings and the primary vesicle is divided into a central pharyngeal chamber and two lateral atrial chambers (Berrill, 1947). Evaginations of the dorsal side of the bud form the neural complex and two posterior evaginations form the origins of the heart and digestive tube (Berrill, 1947). Once a new bud generation is formed, the preceding parental generation is resorbed by apoptosis and phagocytosis (Rinkevich et al., 2013). The process that describes the formation of a new bud and the consequent absorption of the parental zooid is formally referred to as Blastogenesis (Berrill, 1947) (Figure 15). The cycle of zooid formation occurs continuously, resulting in the production of multiple generations per year (Carver, Mallet & Vercaemer, 2006). 
Figure 15

Whole-body regeneration

Botrylloides spp. are capable of self-healing, whereby a lesion is rapidly closed and the remnants of the damaged vascular system are rearranged around the surviving zooids (Lemaire & Piette, 2005; Blanchoud et al., 2018). Similarly, species such as B. lenis exhibit vascular budding, a process that is mediated by the aggregation of lymphocytes which enables the formation of new buds from the vascular epithelium (Oka & Watanabe, 1959).

Members of this genus are capable of an even more dramatic healing phenomenon that is not seen in any other representative of the phylum Chordata (Blanchoud et al., 2018). Botrylloides spp. are capable of developing an entirely new zooid simply from the peripheral vasculature of the original colony (Blanchoud et al., 2018) (Figure 16). This process of whole-body regeneration (WBR) can be inititated by a fragment of the vasculature consisting of as few as 100-200 blood cells (Blanchoud et al., 2018). Recent studies have focused on the mechanisms responsible for WBR and have revealed that stem cells are particularly important in the process (Blanchoud et al., 2018). In species such as B. leachii (Figure 17) and B. violaceus, WBR only occurs when the original colony is reduced to a mass of tissue devoid of any zooids (Blanchoud et al., 2018). In B. leachii colonies, a single zooid is formed by the process of WBR (Figure 17), while in B. violaceus colonies, several zooids may appear. Piwi-positive (Piwi+) cells have been identified as key drivers of WBR in B. violaceus and B. leachii (Oka & Watanabe, 1959; Blanchoud et al., 2018). New insights suggest that genes involved in WBR harbour diverse capabilities and are adopted in a range of bological processes (Blanchoud et al., 2018). However, the mechanisms that drive WBR seem to differ among species as Piwi+ cells are absent during regeneration in Botryllus schlosseri (Pallas, 1766), a close relative of Botrylloides spp. Successful regeneration in B. schlosseri colonies also disparately requires that zooids are removed at a particular stage in blastogenesis and that blood flow through the vasculature remains intact (Blanchoud et al., 2018).

Figure 16
Figure 17


Botrylloides spp. can form chimeras, whereby colonies fuse and act as a single super-organism (Paz & Rinkevich, 2002). Colonies fuse if they share at least one allele in common at a particular polymorphic haplotype (a set of alleles on a single chromosome) (Paz & Rinkevich, 2002). (Allogeneic fusions lead to the resorption of one of the colonies so that the cells of one partner control the entire entity (Paz & Rinkevich, 2002). Botrylloides spp. have also been known to form multiple-partner chimeras (MC), whereby several colonies fuse into a single organism (Paz & Rinkevich, 2002). The formation of chimeras is thought to result in greater energy efficiency as a result of increased colony size (Paz & Rinkevich, 2002). 

The fusion process occurs in the
 following manner: (1) Fusion is initiated when the ampullae of the two colonies interact and form megaloampullae (Figure 18). (2) Hemocytes (blood cells) aggregate in the ampullar tips, and the tunic cuticles begin to disappear where the tunics of the opposing colonies are in contact. (3) The cuticles of the opposing colonies fuse and form a continous tunic matrix. (3) The ampullar tips and the epithelial layers of the colonies merge at the point of contact. At this stage the colonies are entirely fused and a single interconnected vascular system is established. The entire process culminates within 24-48 hrs and appears to occur in a similar manner among Botrylloides spp. (Hirose, 2003; Zaniolo, Manni & Ballarin, 2006). 

The nature of the reaction between colonies is dependent on the type of contact and species combination (Hirose et al., 2002). When incompatible colonies come into contact, rejection reactions are initiated. Contact between the growing edges of incompatible xenogeneic (different species) and allogeneic (same species) colonies can result in the partial fusion of the tunics and the formation of a visible lesion between the colonies (Hirose et al., 2002) (Figure 19). In this instance, hemocytes (mainly morula cells) from the ampullae aggregate at the site of tunic fusion. The cells expel their contents, which react with the surrounding tunic to form the lesion (Hirose, 2003). In some cases, no reaction occurs and no connections are formed between juxtaposed colonies (Hirose et al., 2002). In contrast, contact between cut surfaces of incompatible xenogeneic colonies results in rejection and the formation of a black line along the contact border. When incompatible allogeneic colonies come into contact at their cut surfaces, rejection usually ensues, however fusion has been observed under certain conditions. This 'surgical' fusion has only be observed among viviparous Botrylloides spp., and is thought to result from the absence of allo-reactivity in the vascular system of the colonies (Hirose et al., 2002). The loss of alloreactivity in these viviparous species may be a consequence of prolonged brooding periods as an active allorecognition system might lead to the rejection or destruction of the embryos (Hirose, 2003). 

It has been proposed that the onset of rejection reactions between unrelated colonies is another mechanism by which botryllids prevent overgrowth by competitors and succeed as invaders (Lambert, 2005). 
Figure 18
Figure 19

Anatomy and Physiology


The tunic is perfused with blood vessels and composed of carbohydrates, proteins and tunicin fibers (Monniot, Monniot & Laboute, 1991). An epithelial membrance lies beneath the tunic and separates the visceral organs from the external seawater (Berrill, 1947). This epithelial membrane, along with the cells within the tunic, secrete the products required to form the tunic and enable its continual growth (Monniot, Monniot & Laboute, 1991). The tunic provides structural support to the organism and in some cases protection against predation (Monniot, Monniot & Laboute, 1991). 

Respiratory system

Ascidians of the order Stolidobranchia potentially accumulate iron in their blood (McCabe, 1986). However, assays have revealed that ascidian blood does not have a gas carrying capacity sufficient for life, thus gas exchange occurs via diffusion across the body wall (McCabe, 1986). The large surface area of the pharyngeal basket, the sessile nature of these organisms and the large volume of water being pumped through the animal explain the absence of a sophisticated respiratory system (Ruppert et al., 2004). 

Digestive system

Ascidians bear a looped digestive system. The stomach possesses longitudinal hepatic folds and connects to the intestine (Berrill, 1947). Waste products pass through the intestine and into the anus, which connects to the atrium of the excurrent siphon (Brusca, Moore & Shuster, 2016). Additionally, ascidians have secondarily lost their nephridia, so waste products must be ejected through the excurrent siphon or across the body wall via diffusion (Ruppert et al., 2004). Waste products such as ammonia are most likely expelled via diffusion across the pharynx, whereas urates (uric acid and calcium oxalate) are stored for the lifetime of the zooid (Ruppert et al., 2004). Each zooid possesses an atrial opening, through which the outgoing current is expelled. However, in Botrylloides spp. the atrial openings of adjacent zooids discharge into a common space (Figure 20) so that the water exits through a common excurrent siphon, which helps to concentrate the force of the current (Berrill, 1947). Waste products and larvae can consequently be dispersed efficiently from the main colony (Brusca, Moore & Shuster, 2016). 

Figure 20

Circulatory system

Each botryllid adult zooid possesses a compact heart that lies close to the base of the endostyle (Berrill, 1947). The heartbeat is controlled by two myogenic pacemakers and the direction of blood flow is occasionally reversed (Brusca, Moore & Shuster, 2016). The reversals do not occur synchronously across all zooids of the same colony (Mukai, Sugimoto & Taneda, 1978). The organs and tissues are arranged in series along the circulatory pathway. Therefore, the periodic reversals counterbalance the supply of blood to various tissues by continuously reversing the order in which they receive blood (Ruppert et al., 2004). The colony shares a common circulatory system that is derived from the rudimentary ampullae of the original larva that established the colony (Mukai, Sugimoto & Taneda, 1978). The ampullae extend radially and those that remain connected to the oozooid form connecting vessels that bear sphincters (Figure 21). Upon the degeneration of the zooid, these connecting vessels also disappear, but the peduncles and ampullae remain (Berrill, 1947) (Figure 21). The ampullae of the resorbed oozoid associate with vessels established by subsequent blastozooids (Berrill, 1947). In established colonies, the ampullae of several zooids are mostly concentrated along the outer margin of the colony (Mukai, Sugimoto & Taneda, 1978). The ampullae pump blood throughout the colony via autonomous contractions and expansions which occur at regular intervals (Newberry, 1964; Burighel & Brunetti, 1971). Hemocytes and nutrients are shared between zooids within this colonial vascular system (Gutierrez & Brown, 2017).
Figure 21

Reproductive system

In Botrylloides spp. the testis and egg/s mature on either side of the zooid in the mantle. The ovary is positioned posterior to the testis, a feature unique to this genus (Rocha et al., 2019) (Figure 3). The egg is encased in two follicular layers and attached to the atrial brood pouch (Figure 11) by a vesicular follicle stalk (Mukai, Saito & Watanabe, 1987). The pouch arises as a thickening of atrial (peribranchial wall) epithelium during bud development and forms adjacent to the ovary (Mukai, Saito & Watanabe, 1987) (Figure 11). The egg is ovulated, fertilised and undergoes embryogenesis within the brood pouch (Mukai, Saito & Watanabe, 1987).  

Nervous system

The nervous system of species from the genus Botrylloides has not been studied extensively. However, work has been done on a related species, Botryllus schlosseri. In B. schlosseri the embryonic nervous system differentiates from the neural plate and the brain exhibits tripartite organisation (Monniot, Monniot & Laboute, 1991). During metamorphosis, the larval nervous system is replaced by a new nervous system in the developing oozoid (Figure 22). The adult zooid possesses a cerebral ganglion (brain) connected to a peripheral nervous plexus and adjacent to a neural gland (Monniot, Monniot & Laboute, 1991; Sawada, Yokosawa & Lambert, 2001) (Figure 23). The cerebral ganglion is derived from neuroblasts that originated in the neural gland (Monniot, Monniot & Laboute, 1991; Zaniolo et al., 2002). The neural gland arises from the larval anterior neural tube and is comprised of a cluster of cells (Monniot, Monniot & Laboute, 1991; Zaniolo et al., 2002). Nerves from the brain innervate the atrial and oral (buccal) siphons, the stigmata and longitudinal muscles of the body wall (Markman, 1958). A dorsal cord, which innervates the gut and endostyle, runs between the gonads and the neural gland (Markman, 1958) (Figure 23). Nerves do not seem to connect zooids within a colony, thus synchronous behaviour may be controlled by the tunic epithelium. The epithelium responds to action potentials transmitted between cells that communicate at gap junctions (Zaniolo et al., 2002). The sensory cells in ascidians have been investigated and seem to respond to light, chemical and physical stimuli (Monniot, Monniot & Laboute, 1991).

Ascidian larvae have a vastly different central nervous system to that of the adult stage, as it is adapted to their pelagic lifestyle. An anterior ganglion and a sensory vesicle are found in the trunk of the larva (see 'Sexual reproduction' section). The dorsal nerve cord and notochord are homologous to those seen in vertebrates (Monniot, Monniot & Laboute, 1991) (see 'Evolution' subsection). 
Figure 22
Figure 23

Biogeographic Distribution

Global distribution

The most important factors mediating the biogeographic distribution of ascidians are temperature, salinity, hydrodynamics and light (Lambert, 2005). Considering this, representatives of the genus Botrylloides are widely distributed and are found globally (Table 2). Species that are the most tolerant of adverse conditions, such as warming sea temperatures, often exhibit the greatest potential to colonise novel environments (Lambert, 2003).

Table 2: A summary of the entire geographic range of species belonging to the genus Botrylloides that have been observed in Australian waters (Kott, 1952; Kott 1985; Kott, 2005). Since these early observations, it would seem that B. magnicoecum and B. niger were incorrectly identified in Australia (Atlas of living Australia, 2020). 


Distribution outside of Australia

Distribution in Australian waters

B. violaceus (Oka, 1927)

North Pacific, Western Pacific and Japan

New South Wales, Queensland and Western Australia

B. leachii (Savigny, 1816)

Red Sea, North-east Atlantic and Mediterranean

Western Australia, South Australia, Victoria and New South Wales

B. anceps (Herdman, 1891)

North-east Atlantic, North Sea, Black Sea, Adriatic Sea, tropical Indo-west Pacific, Mediterranean Sea and Red Sea  

South Australia, Tasmania, Victoria and Western Australia

B. saccus (Kott, 2003)


South Australia

B. perspicuus (Herdman, 1886)

Philippines, Indonesia and Hong Kong (Kott, 2005)

South Australia, Western Australia, Tasmania, Victoria, New South Wales and Queensland

B. magnicoecum (Hartmeyer, 1912)

China, West Indian Ocean, South Africa, West Africa and New Zealand 

Victoria, Western Australia, South Australia, Tasmania, Victoria, New South Wales and Queensland

B. niger (Herdman, 1886)

Malaya, New Zealand, South Africa, Mediterranean, North-west Europe and North Atlantic

Completely cosmopolitan around Australia

Australian distribution

The species B. violaceus, B. leachii, B. anceps, B. saccus and B. perspicuus have all been recently sighted in Australian waters (Kott, 2005; Atlas of living Australia, 2020) (Figures 24 & 25). B. violaceus is a particular concern as it is currently considered globally invasive (Viard et al., 2019). Most of these species were first documented and identified in Australian waters by Kott based on morphological and anatomical features (1952; 1985) (Figure 25). However, taxonomic allocations and species occurences are continously being updated (see 'Species diversity' section). 
Figure 24
Figure 25

Evolution and Systematics


Phylum: Chordata (Haeckel, 1874)
Subphylum: Tunicata (Lamarck, 1816)
Class: Ascidiacea (Blainville, 1824)
Order: Stolidobranchia (Lahille, 1886)
Family: Styelidae (Sluiter, 1895)
Genus: Botrylloides (Milne Edwards, 1841)

The family Styelidae (Figure 26) is composed of 38 genera and 535 known species (Shenkar & Swalla, 2011). Members of this family bear four pharyngeal folds on either side of the body, branchial tentacles, at least one gonad, longitudinal muscles overlaid with circular muscles, and some endocarps (raised portions of the internal parietal body wall) (Kott, 1985).The botryllids are difficult to distinguish in the field. However, the position of the ovaries and the presence of a brood pouch are usually used to assign species to a genus (Rocha et al., 2019) (list below). Some species from the genus Botryllus exhibit features that are characteristic of Botrylloides spp. For example, Botryllus delicatus and Botryllus sexiens possess a sac-like brooding organ analogous to that of Botrylloides. Yet, the ovary is anterior to the testis in their zooids, characteristic of the genus Botryllus (Okuyama & Saito, 2001). Consequently, some studies have suggested that the origin of the brooding organ should be considered during classification (Okuyama & Saito, 2001) (Table 3). In Botrylloides spp., the brooding organ originates from the peribranchial epithelium that is adjacent to the ovaries (Figure 11). This allows information to transfer more easily between the peribranchial epithelium and the mature oocyte (Okuyama & Saito, 2001). 

A summary of the characteristics used to identify Botrylloides spp. are as follows (Sawada, Yokosawa & Lambert, 2001): (1) zooids do not possess individual atrial siphons, (2) zooids are arranged in meandering or ladder-like systems, (3) the ovary is placed posterior to the testis, (4) the brood pouch arises from the peribranchial epithelium adjacent to the ovaries, and (5) species may be viviparous or ovoviviparous.

Figure 26

Species diversity

There are currently 18 confirmed species of the genus Botrylloides (Shenkar et al., 2016). Species from this genus appear morphologically similar and as research findings progress, several species have been revealed to be duplicates of each other in certain regions. For example, B. giganteum (Peres, 1949) was recently confirmed to be synonymous with B. pizoni (Brunetti & Mastrototaro, 2012) (Rocha et al., 2019). In other cases, morphologically indistinguishable forms have been established as distinct species such as B. perspicuus and B. giganteum (Rocha et al., 2019). New species are also continuously being identified as our molecular tools evolve (Figure 27). This is exemplified by the recent establishment of Botrylloides conchyliatus sp. in 2019 based on morphological and genetic analyses (barcoding using the cytochrome c oxidase subunit I (COI) gene) (Rocha et al., 2019). Detailed molecular studies (such as barcoding) are increasingly used to reliably identify species in this genus (Viard et al., 2019). 
Figure 27


Typical chordate features are present in the larval form of the Ascidiacea, including a pharynx with gill slits, a notochord, a dorsal nerve cord and muscle segments (Miller, 1971; Satoh, 2003) (Figure 12). Within the Chordata, the Tunicata are a particulary interesting group because they are likely to be the closest extant relatives of the Vertebrata (Figure 25). The chordate subphyla Vertebrata and Tunicata share several synapomorphies. For example, the oral opening and the anterior neural plate territories in amphibians and ascidians share a similar origin. Secondly, the sensory placodes in ascidians appear to be related to the sensory neural structures present in vertebrates (Lemaire & Piette, 2015). The tissue complexity of ascidians is also similar to that of vertebrates and both taxa possess a heart, notochord, endostyle and vascular system (Blanchoud et al., 2018). 

The tunicate order Stolidobranchia has been at the forefront of recent evolutionary study as its representatives exhibit remarkable biological processes, many of which have been discussed on this webpage. There is evidence to suggest that genome characteristics apparent in the Stolidobranchia group, such as transposon diversity, genome size and gene number, may contribute to their success as invaders (Blanchoud et al., 2018). Genes identified in B. schlosseri and B. leachii appear to impart biological processes unique to colonial tunicates. These processes include wound healing, circulation, cell communication and immune response (Blanchoud et al., 2018). The genes responsible for these processes have orthologs in vertebrates and more ancient taxa such as Caenorhabditi elegans. This infers that these genes share a very ancient origin and were retained in the botryllids (Blanchoud et al., 2018). Futher research on this taxon is expected to yield insights that will greatly advance the fields of medicine and immunology. 

Conservation and Threats

Insufficient water flow and high siltation could negatively impact colonies as they rely on filter feeding (Bak et al., 1998; Bone, Carre & Chang, 2003; Carver, Mallet & Vercaemer, 2006). Studies have found that Botrylloides spp. are particularly sensitive to synthetic chemical compounds. For example, exposure to Organotin compounds (OTC), which are incorporated in fungicides, pesticides and anti-fouling agents, results in embryotoxicity and immunotoxicity (Sawada, Yokosawa & Lambert, 2001). This causes delayed embryonic and larval development, and the impairment of immune system components, respectively (Sawada, Yokosawa & Lambert, 2001). Most significantly, OTC hinders Ca2+ homeostasis in immunocytes (immune cells) which results in changes to cell shape, motility and even apoptosis (Sawada, Yokosawa & Lambert, 2001). These findings suggest that Botrylloides spp. may be adversely affected by other foreign chemicals that infiltrate marine environments as a result of anthropogenic activities. 

Most studies have focused on the negative invasive potential of this genus and developing potential mitigation strategies. Very little work has been done to determine the environmental threats to botryllid ascidians as they appear to be extremely tolerant of a wide range of conditions, hence their success as dominant biofoulers (Lambert, 2005). It should be a cautionary note in terms of environmental impact that successful antifouling methods and controls could significantly diminish populations of this intriguing group of ascidians. Control and prevention should be monitored for excessive adverse impact. 


Atlas of living Australia. (2020). Botrylloides Milne-Edwards, 1841. Retrieved May 22, 2020, from  

Bak, R. P. M., Joenje, M., de Jong, I., Lambrechts, D. Y. M., & Nieuwland, G. (1998). Bacterial suspension feeding by coral reef benthic organisms. Marine Ecology Progress Series, 175, 285–288.

Berrill, N. J. (1947). The developmental cycle of BotrylloidesThe Quarterly journal of microscopical science, 88(4), 393.

Berrill, M. (1971). Developmental biology. New York: McGraw-Hill. 

Bishop, J. D. D. (1996). Female control of paternity in the internally fertilizing compound ascidian Diplosoma listerianum. Autoradiographic investigation of sperm movements in the female reproductive tract. Proceedings of the Royal Society of London. Series B: Biological Sciences, 263(1368), 369-376. doi: 10.1098/rspb.1996.0057

Blanchoud, S., Rutherford, K., Zondag, L., Gemmell, N. J., & Wilson, M. J. (2018). De novo draft assembly of theBotrylloides leachii genome provides further insight into tunicate evolution. Scientific Reports, 8(1), 5518. doi: 10.1038/s41598-018-23749-w

Blanchoud, S., Zondag, L., Lamare, M. D., & Wilson, M. J. (2017). Hematological Analysis of the Ascidian Botrylloides leachii (Savigny, 1816) During Whole-Body Regeneration. The Biological Bulletin, 232(3),143-157. doi: 10.1086/692841

Bock, D. G., Zhan, A., Lejeusne, C., MacIsaac, H. J., & Cristescu, M. E. (2011). Looking at both sides of the invasion: patterns of colonization in the violet tunicate Botrylloides violaceusMolecular Ecology, 20, 503–516.

Bone, Q., Carre, C., & Chang, P. (2003). Tunicate feeding filters. Journal Marine Biological Association of the United Kingdom, 83, 907–919. 

Brusca, R. C., Moore, W., & Shuster, S. M. (2016). Invertebrates (3rd ed.). Sunderland, Massachusetts: Sinauer Associates.

Bullard, S., Sedlack, B., Reinhardt, J., Litty, C., Gareau, K., & Whitlatch, R. (2007). Fragmentation of colonial ascidians: Differences in reattachment capability among species. Journal of Experimental Marine Biology and Ecology, 342, 166-168. doi: 10.1016/j.jembe.2006.10.034

Burighel., P., & Brunetti, R. (1971). The circulatory system in the blastozooid of the colonial ascidian Botryllus schlosseri(Pallas). Bollettino di zoologia, 38, 273-289.

Carver, C., Mallet, A., & Vercaemer, B. (2005). Biological Synopsis of the colonial tunicates, Botryllus schlosseri andBotrylloides violaceusCanadian Manuscript Report of Fisheries and Aquatic Science, 2747.

Gutierrez, S., & Brown, F. D. (2017). Vascular budding in Symplegma brakenhielmi and the evolution of coloniality in styelid ascidians. Developmental Biology423(2), 152-169.

Hirose, E. (2003). Colonial Allorecognition, Hemolytic Rejection, and Viviparity in Botryllid Ascidians. Zoological Science, 20(4), 387-394. doi: 10.2108/zsj.20.387

Hirose, E., Shirae, M., & Saito, Y. (2002). Colony specificity in the xenogeneic combinations among fourBotrylloides species (urochordata, ascidiacea). Zoological Science, 19(7), 747. doi: 10.2108/zsj.19.747

Holland, L. Z. (2016). Tunicates. Current Biology, 26(4), 146-152. doi:

Hughes, D., Cook, E., & Sayer, M. (2005). Biofiltration and biofouling on artificial structures in Europe: The potential for mitigating organic impacts. Oceanography and Marine Biology, 43, 123-172.

Hyams, Y., Paz, G., Rabinowitz, C., & Rinkevich, B. (2017). Insights into the unique torpor of Botrylloides leachi, a colonial urochordate. Developmental Biology, 428(1), 101-117. doi: 10.1016/j.ydbio.2017.05.020

Kott, P. (1952). The Ascidians of Australia. I. Stolidobranchiata Lahille and Phlebobranchiata Lahille. Marine and Freshwater Research, 3(3), 205-334. doi: 10.1071/MF9520205

Kott, P. (1985). The Australian Ascidiacea part 1, Phleobranchia and Stolidobranchia. Memoirs of the Queensland Museum, 23, 1-440. 

Kott, P. (2005). Catalogue of Tunicata in Australian waters. Canberra, AustraliaAustralian Biological Resources Study. 

Lambert, G. (2005). Ecology and natural history of the protochordates. Canadian Journal of Zoology, 83(1), 34-50. doi: 10.1139/z04-156

Lemaire, P., & Piette, J. (2015). Tunicates: exploring the sea shores and roaming the open ocean. A tribute to Thomas Huxley. Open Biology, 5(6). doi: 10.1098/rsob.150053

Mackie, G. O., & Burighel, P. (2005). The nervous system in adult tunicates: current research directions. Canadian Journal of Zoology, 83(1), 151-183. doi: 10.1139/z04-177

Markman, B. (1958). On the peripheral nervous system of ascidians. Acta Zoologica, 39(1), 13-18. doi: 10.1111/j.1463-6395.1958.tb00518.x 

McCabe, M. (1986). Respiratory Gas Exchange in Ascidians: An Almost Diffusion Limited Animal with a Cardiovascular System. In: I. S. Longmuir (Ed.), Oxygen Transport to Tissue VIII. Advances in Experimental Medicine and Biology (vol. 200). Boston, MA: Springer. 

Milkman, R. (1967). Genetic and developmental studies on Botryllus schlosseri. Biological Bulletin, 132, 229-243.

Millar, R. H. (1971). The biology of ascidians. Advances in Marine Biology, 9, 1-100

Monniot, C., Monniot, F., & Laboute, P. (1991). Coral Reef Ascidians of New Caledonia. Paris, France: ORSTOM.

Mukai, H., Sugimoto, K., & Taneda, Y. (1978). Comparative studies on the circulatory system of the compound ascidians,Botryllus, Botrylloides and SymplegmaJournal of Morphology, 157(1), 49-77. doi: 10.1002/jmor.1051570105

Newberry, A. T. (1965). The structure of the circulatory supports of the test and its role in budding in the Polystyelid ascidian Metandrocarpa tayloriAcademie Royale de Belgique Classe des Sciences Memoires Collection in 4 Deuxieme serie, 16, 1-67.

Oka, H., & Watanabe, H. (1959). Vascular budding in BotrylloidesThe Biological Bulletin, 117(2), 340-346. doi: 10.2307/1538913

Okuyama, M., & Saito, Y. (2001). Studies on the Structure of the Brooding Organs of Two Botryllid Ascidians, Botryllus delicatus and Botryllus sexiens. Zoological Science, 18(3), 397-404. doi: 10.2108/zjs.18.397

Paz, G., & Rinkevich, B. (2002). Morphological consequences for multi-partner chimerism in Botrylloides, a clonial urochordate. Developmental and Comparative Immunology, 26(7), 615-622. 

Pisut, D. P., & Pawlik, J. R. (2002). Anti-predatory chemical defenses of ascidians: secondary metabolites or inorganic acids? Journal of Experimental Marine Biology and Ecology, 270(2), 203-214. doi: 10.1016/S0022-0981(02)00023-0

Reem, E., Douek, J., & Rinkevich, B. (2018). Ambiguities in the taxonomic assignment and species delineation of botryllid ascidians from the Israeli Mediterranean and other coastlines. Mitochondrial DNA Part A, 29(7), 1073-1080. doi: 10.1080/24701394.2017.1404047

Rinkevich, B., Shashar, N., & Liberman, T. (1993). Nontransitive xenogeneic interactions between four common Red Sea sessile invertebrates. Proceedings of the Seventh International Coral Reef Symposium, 2, 833-839. 

Rinkevich, Y., Voskoboynik, A., Rosner, A., Rabinowitz, C., Paz, G., Oren, M., . . . Rinkevich, B. (2013). Repeated, Long-Term Cycling of Putative Stem Cells between Niches in a Basal Chordate. Developmental Cell, 24(1), 76-88. doi: 10.1016/J.devcel.2012.11.010

Rocha, R. M., Salonna, M., Griggio, F.,Ekins, M., Lambert, G., Mastrototaro, F., . . . Gissi, C. (2019). The power ofcombined molecular and morphological analyses for the genus Botrylloides: identification of a potentially global invasive ascidian and description of a new species. Systematics and Biodiversity, 17(5), 509-526. doi: 10.1080/14772000.2019.1649738 

Rodriguez, D., Kassmer, S. H., & De Tomaso, A. W. (2017). Gonad development and hermaphroditism in the ascidian Botryllus schlosseri. Molecular Reproduction and Development, 84(2), 158-170. doi: 10.1002/mrd.22661

Ruppert, E. E., Barnes, R.D., & Fox, R.S. (2004). Tunicata (Urochordata). Invertebrate zoology: A functional evolutionary appoach (7th ed.). California: Brooks/Cole.

Sabbadin, A. (1955). Osservazioni sullosviluppo, l'accrescimento e la riproduzione di Botryllus schlosseri (Pallas) in condizioni di laboratorio. Bollettino di zoologia, 22, 243-263. 

Saito, Y., & Watanabe, H. (1985). Studies on Japanese compound styelid ascidians 4. Three new species of the genus Botrylloides from the vicinity of Shimoda. Publications of the Seto Marine Biological Laboratory, 30(4-6), 227-240. doi: 10.5134/176112

Satoh, N. (2003). The ascidian tadpole larva: comparative molecular development and genomics. Nature Reviews Genetics, 4(4), 285-295. doi: 10.1038/nrg1042

Sawada, H., Yokosawa, H., & Lambert, C. C. (2001). The Biology of Ascidians (1st ed.). Japan, Tokyo: Springer. 

Shenkar, N., & Swalla, B. J. (2011). Global Diversity of Ascidiacea (Review). PLoS ONE, 6(6), e20657. doi: 10.1371/journal.pone.0020657

Shenkar, N., Gittenberger, A., Lambert, G., Rius, M., Moreira da Rocha, R., Swalla, B. J., & Turon, X. (2020). Ascidiacea World Database. Retrieved May 10, 2020, from 

Simkanin, C., Dower, J. F., Filip, N., Jamieson, G., & Therriault, T.W. (2013). Biotic resistance to the infiltration of natural benthic habitats: examining the role of predation in the distribution of the invasive ascidian Botrylloides violaceusJournal of Experimental Marine Biology and Ecology, 439, 76-83.

Sorrentino, M., Manni, L., Lane, N. J., & Burighel, P. (2000). Evolution of cerebral vesicles and their sensory organs in an ascidian larva. Acta Zoologica, 81(3), 243-258.doi: 10.1046/j.1463-6395.2000.00054.x

Stabili, L., Licciano, M., Gravina, M. F., & Giangrande, A. (2016). Filtering activity on a pure culture of Vibrio alginolyticus by the solitary ascidian Styela plicata and the colonial ascidian Polyandrocarpa zorritensis: a potential service to improve microbiological seawater quality economically. Science of The Total Environment, 573, 11-18.

Svane, I., & Young, C. M. (1989). The ecology and behaviour of ascidian larvae. Oceanography and Marine Biology: an Annual Review, 27, 45-90.

Tarjuelo, I., Lopez-Legentil, S., Codina, M., & Turon, X. (2002). Defence mechanisms of adults and larvae of colonial ascidians: Patterns of palatability and toxicity. Marine Ecology Progress Series, 235, 103-115. doi: 10.3354/m

Viard, F., Roby, C., Turon, X., Bouchemousse, S., & Bishop, J. (2019). Cryptic Diversity and Database Errors Challenge Non-indigenous Species Surveys: An Illustration With Botrylloides spp. in the English Channel and Mediterranean Sea.Frontiers in Marine Science, 6(615). doi: 10.3389/fmars.2019.00615

Westerman, E. (2007). Impacts of changing water temperatures on the life histories of two invasive ascidians in the Gulf of Maine: Botryllus schlosseri and Botrylloides violaceus. In L. G. Harris & J. A. Bolker (Eds.): ProQuest Dissertations Publishing.

Zaniolo, G., Lane, N. J., Burighel, P., & Manni, L. (2002). Development of the motor nervous system in ascidians. Journal of Comparative Neurology, 443(2), 124-135. doi: 10.1002/cne.10097

Zaniolo, G. L., Manni, L., & Ballarin, L. (2006). Colony specificity in Botrylloides leachi. I. Morphological aspects.Invertebrate Survival Journal, 3. 

Zaniolo, G., Manni, L., Brunetti, R., & Burighel, P. (1998). Brood pouch differentiation in Botrylloides violaceus, a viviparous ascidian (Tunicata). Invertebrate Reproduction & Development, 33(1), 11-23. doi: 10.1080/07924259.1998.9652338


The author would like to thank all those who helped deploy and retrieve the settlement plates from MB. I would also like to thank the BIOL3211 tutors and most importantly Sandie and Bernie Degnan for their guidance. Lastly, I would like to acknowledge the contribution of Professor Billie Swalla who shared some invaluable insights and advice with me.