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Stolons and Non-Embryonic Development in Colonial Ascidians

Tallulah Southby Osborne 2020


The Urochordata class, Ascidiaecea, consists of a large diversity of morphologies and reproductive strategies that allow these animals to colonise and grow quickly across a wide variety of marine habitats. They are apart of the Tunicata due to the external ‘tunic’ excreted during the sessile adult phase (Holland, 2016). Colonial ascidians are large colonies of connected, genetically identical, zooids. These are often split into two categories: social and colonial, based on whether the zooids exist within a shared test or as separate individuals connected by a vascular system. Some aggregations of solitary ascidians can appear to work as social ascidians in terms of spatial colonisation, and genetically identical zooids have been known to fuse through allorecognition (Pérez-Portela et al., 2016; Saito, Hirose & Watanabe, 2002). It has been proposed that the presence or absence of a shared vascular system should be used instead (Pérez-Portela et al., 2016).

Stolons are vascularised tissue that connect units within most sessile colonial organisms, including plants, fungi and marine invertebrates (Buss, 1979). They have been described in terms of two general growth patterns: ‘guerrilla’, which consists of longer connecting stolons (or runners) that allow the organism to scout suitable location within its settled environment, and ‘phalanx’ which consists of shorter stolons, and more dense repeating individuals that secure their space and settle (Vogt et al., 2011). These terms are usually used for plants but have been used to describe the growth and colonising patterns of marine invertebrates, namely hydroids. Vogt et al. (2011) examined stolons in colonial hydroids, describing “sheet-like” and “runner-like” morphologies, discussing how stolons are used by colonies to be able to respond to environmental change. By connecting the individual hydroid polyps, stolons allow nutrients to be transported amongst them. The polyps can continue to function if separated from the colony, giving them morphological plasticity to respond to disturbances and allow for dispersal (Vogt et al., 2011).

Ascidians are the only chordates with regenerative abilities (Tiozzo et al., 2008), and colonial ascidians utilise stem cells, asexual budding and non-embryonic development to colonise space. In social ascidians, it’s often vascular or stolonic budding that facilitates this. Given the similarities of colonial hydroids and colonial ascidians in their trophic levels and ecological interactions within marine benthic communities, parallels may be drawn between the extensive research on stolon development and function in hydroids to colonial ascidians. There is less research on the stolons of social ascidians, which may be due to the enormous diversity of body morphs and budding systems.

Physical Description

Patricia Kott (2001), who specialised in ascidians working with the Queensland Museum, discussed the difficulties in describing colonies as factors such as age, temperature and oxidation can change the colour, shape, boundary and preservation of ascidian colonial systems.

Social ascidians are found in all three orders. Rather than being set in a shared test like compound colonial ascidians, social ascidians make colonies consisting of individual zooids connected by stolons or other vascular connective tissues (Figure 2.; Ritter, 1897). The zooids themselves have a large variety in body size and distribution and orientation to the sea floor (Kott, 2001). The zooids are genetically identical, which gives these colonies large plasticity through the ability to fuse or regenerate (Saito, Hirose & Watanabe, 2002). Some grow in dense colonies, and others are more widely spaced (Figure X). Stolons are an outward, elongated fold of the mantle containing blood and mesenchyme cells that extends from the base of the zooid (Monniot et al. 1991). Stolons vary in their appearance, and often overlap with generic blood vessels in terms of appearance, making it difficult to distinguish between them. They are most visible in social ascidians as branching structures. An example of a social ascidian from the Styelidae family is Stolonica socialis (Hartmeyer, 1903). It forms dense patches of small zooids that are connected at the base by a branch-like stolon not often visible underneath the dense growth (Ager, 2007). Another example is the Perophera viridis (Verrill, 1871), that has thin green stolons that are tubular and branching (Deviney 1934). They can easily be seen between the more widely spaced zooids (Figure Mb.).

The specimen identified on the ARMS plates suspended in Dunwich Bay has transparent stolons connecting individual zooids from their base. The zooids are 1mm in diameter and height, with extruding siphons on the ventral surface (Figure 1.) Distinct orange ovals are contained within the membrane (Figure 2a.). The stolons vary in length and width. A lack of information about this species growth and internal morphology means that the role of the stolons for this specimen can’t be concluded. However, based on the external morphology it is hypothesised that the stolons are vascularised, and are transporting nutrients, cells and stem cells between the zooids. They also play a large role in asexual budding (see Life History for more information).

Brown and Swalla (2012) distinguish between stolonic budding and vascular budding as vascular consist of sporadic and random growth, whereas stolonic appears to occur at regular intervals. Once considering the difference in the role of the stolon within budding and different ascidian families, the connective tissue seen on the unidentified ARMS plate specimen may show connective vessels instead of stolons. The vessels contain blood cells that are being pumped from the parent zooid to undergo vascular budding (Figure 2). Stolons are often identified as narrower, with swelling that occurs at nodes. It is difficult to distinguish between the budding modes from photographs alone, but it appears that the zooids in the specimen bud at random intervals (Figure 2c.).
Figure 1
Figure 2


Many sessile and sedentary organisms have developed colonial forms which increase their competitive ability over solitary organisms (Jackson, 1977). Solitary organisms are limited by their surface area to size ratio which impacts how quickly they can grow. Colonial organisms eliminate this problem as they increase surface area more rapidly with individual zooids that are sharing the resources gained from the surrounding environment (Burgess et al., 2017). They can grow faster and also reproduce more quickly via budding than solitary individuals (Jackson & Coates, 1986; Hughes, 1989). Social ascidians are an important part of settlement and benthic communities.

Social ascidians can occur in dense colonies or larger branching communities meaning they add competitive pressure for surface space on other sessile and sedentary organisms around them, which is often a limited resource in rocky and reef environments. The branching nature of the stolons allows colonial organisms to rapidly claim space when they first settle. By spreading the stolons wide from the first settled larvae, space is claimed from other settling organisms as zooids form via vascular and stolonic budding (Alié et al., 2018).

A stalked stolon is seen in some species of the Pyuridae and Styelidae genera (Kott, 1985). This allows the body of the ascidian to be lifted above the substrate, exploiting the substratum niche (see Figure X; Young & Braithwaite, 1980). This gives those species a competitive advantage for food as they filter water and compete less for space than their flat relatives (Kott, 1985). In this case, the stolon often has adhesive properties so it can act as an anchor to the surface (Kott, 1985; Li et al., 2019). In colonial stalked species, it also acts as a vessel for nutrient transport, allowing individual zooids to effectively avoid the benthic competition for food while simultaneously sharing the nutrients filtered.

The specimen observed grew on algal covered surfaces, and had silt settled on the stolons (Figure 3). However, no other species grew over it which means it may have settled after the surrounding sponges and bryozoans or it has a defence mechanism that allows it to successfully compete for space.
Figure 3

Life History and Behaviour

Sexual Reproduction

Most colonial ascidians reproduce sexually and asexually. Most hermaphroditic and use broadcast spawning when sexually reproducing. The embryos brood within the parent and when developed, the larvae will disperse, allowing the colony to spread widely (Sawada et al., 2001). Of interest for socially colonial (and compound colonial) ascidians is asexual reproduction. Asexual reproduction through budding and regeneration allows them a robustness to environmental disturbances, as well as the ability for rapid colonisation (Kurn et al. 2011). Once colonial ascidians metamorphose to their adult state, budding occurs their whole lives as a form of colony growth. In social species with external vasculature, the zooids can reproduce sexually or asexually. Sexual reproduction would aid in propagation of the colony and allow for it to disperse. Asexual budding allows the parent colony to continue to grow (Kurn et al. 2011; Sawada et al. 2001).

Colonising Strategies

Colonising strategies are important for ascidians in order to develop and successfully compete for light, nutrients and space. Majority of research on colonising behaviour in ascidians has been done on the colonial Botrylloides, a genus where the individual zooids share a test and asexually reproduce via budding.

The specimen seen on the ARMS plates (Figures 1 and 2) is very small and seems to be spaced quite sparsely. This could be because it may be a young colony and the zooids are still growing. It could also be a colonising technique – to establish an individual zooid at a distance from the last. Budding could then occur along the length of the stolons as the zooids gain more nutrients and grow more, eventually leading to a more dense patch of individuals. (Refer to Non-embryonic development).

An experiment conducted by Sugino and Nakauchi (1987) showed that within the colonial species Symplegma reptans (Oka, 1927), regeneration and budding are processes controlled on the individual and colony level. This species belongs to the Styelidae family, where blastogenic and vascular budding have been described as methods of asexual reproduction arising in the multiple times that coloniality has developed within Stolidobranchs. The role of ambient zooids within the same colony was highlighted as inhibiting budding; when buds were removed at the oozoid stage, they found that the adults grew twice as many buds. Although specific to this species, similar results have been found in other research (Sugino & Nakauchi, 1987; Gutierrez & Brown, 2017), further supporting the importance of asexual budding as a mechanism that allows organisms to persist through regeneration even during the early-development stages.

Non-embryonic Development

(see also Anatomy and Physiology)

Multiple modes of budding have been identified across colonial ascidians (Gutierrez & Brown, 2017; Alié et al., 2018; Hughes, 1989; Monniot et al., 1991). This reflects the plasticity of non-embryonic development in colonial ascidians which allows them to compete for space, disperse and multiply in response to environmental conditions (Scelzo et al., 2019). Table 1 provides an overview of some of the broad range of asexual budding described in the literature.

Turon (2005) proposes a distinction between colony growth and colony multiplication, creating a distinction between propagative budding and multiplicative budding. Propagative budding, a term used by Nakauchi (1987), includes the creation of a new generation of colonies through blastozooids from an original zooid. Turon suggests that multiplicative budding should encompass budding modes modified from known stolonic and pallial budding seen in some Phlebobranchia and Stolidobranchia species (he specifically refers to Perophera japonica and Polyzoa vesiculophora; Table 1.)

Alié et al. (2018) describe two types of budding within the colonial individuals of the Styelidae family, that have arisen convergently as methods of asexual propagation and regeneration: 1) Peribranchial budding, which occurs from the epithelium of a zooid and, 2) vascular budding, which occurs within the vascular system of the colony (Table 1). Stolonic budding is similar to vascular budding but occurs more frequently in social ascidians. Vascular budding is more common in Botrylloides within the internal connective blood vessels (Alié et al. 2018; Deviney, 1934).

Stolonic Budding

Stolonic budding is studied most in the Perophoridae family. The stolon protruding from the base of the zooid contains mesenchyme cells and blood which aggregate either along the stolon or at the ends, causing a swelling. Hemocytes (see Anatomy and Physiology) cause hernias which then undergo organogenesis which develop into fully formed zooids (Monniot et al. 1991; Brown & Swalla, 2008). The process is very similar to vascular budding, suggesting the adaptation of the mechanism in some species. Scelzo et al. (2019) described how stolonic budding has evolved once in the Botryllids, but modified budding has occurred in other species, such as Polyandrocarpa zorritensis (refer to Figure 4.).

Turon (2005) described a new form of “star-shaped” budding in the Aplousobranchia genus, Clavelina, that appears to have evolved from the stolonic budding (vascular) seen in other members of this order (see Table 1.). Turon’s research shows yet another modification to budding modes, highlighting their importance to the dispersal and colonisation of ascidians. By having developed buds dispersing, this Clavelina species can quickly colonise a new area through stolonic budding, much faster than the settlement and metamorphosis of a solitary ascidian (Turon, 2005; Jackson, 1977; Jackson & Coates, 1986).

Table 1. A summary of the some plasticity in asexual budding that occurs within colonial ascidians across the Ascidiacea taxa. Information compiled from Turon (2005); Alié et al. (2018); Gutierrez and Brown (2017); and Brown and Swalla (2008), Monniot et al., (1991).

Mode of budding Variation (plasticity) Role as defined by Turon (2005) Taxa Mechanism
Propagative Styelidae Bud forms from the peribranchial chamber of the individual zooid, remain attached (Alie et al. 2018)

Transdifferentiation of mesenchymal cells Propagative Polyandrocarpa misakiensis Integration of transdifferentiated mesenchymal into inner vesicle, bud separates from zooid (Scelzo et al. 2019)

Epithelial protrusion Propagative Stolonica socialis The epithelial layer protrudes, and and the peribranchial layer invaginates, creating a double vesicle (Sclezo et al. 2019).
Stolonial/Vascular (refer to Fig.4)
Multiplicative (and regenerative) Styelidae, Perophoridae Proliferation of circulatory (stem) cells in the vasculature of the colony (Alié et al. 2018; Brown & Swalla. 2008; Monniot et al., 1991)

Star budding Propagative and Multiplicative
Budding from the basal blood vessel break-off and disperse. When these buds settle the centre becomes a blastozooid, while the star ‘arms’ form stolons (Turon, 2005).
Palleal (sometimes used interchangeably with peribranchial)
Multiplicative Styelidae The development of terminal ampullae from a vessel within the mantle wall, creating a planktonic bud (Gutierrez and Brown 2017).
Multiplicative and Propagative Aplousobranchia, Polyclinidae The elongated body is segmented and reorganises while growing – the zooids have three parts (Brown & Swalla, 2008; Monniot et al., 1991).
Multiplicative Aplousobranchia Two buds derived from the epicaridnal region, fusing between old and new abdomens creating two individuals (Brown and Swalla, 2008)
Figure 4

Anatomy and Physiology

Hirose and Akahori (2004) compare the stolonic vessel of the Aplousobranchia didemnid species to tissues related in blood vessels that connect zooids in clavelinid species (refer to Fig. 5). Their study highlights the varying tissues that make-up colonial species. Although focusing significantly on the tissue morphology and function, this provides some hints into how species may be related (further genetic analysis would be necessary for stronger conclusions). Within the compared species, it was found that the stolonic vessel does not connect individual zooids as the stolon does, but has similarities in that both contain hemocyte and have a columnar distal tip. The stolon itself has a lateral wall and distal tip made up of columnar cells containing heterogenous vesicles and ends bluntly where the stolonic vessel would bulge (Hirose & Akahori, 2004).

While the role of hemocyte within the non-connective stolonic vessels of didemnid species is relatively unknown (Hirose & Akahori, 2004), the hemocyte found in vasculature of the Botryllids are involved in the regenerative processes of budding (Oka & Watanabe, 1957; Alié et al., 2018; Brown & Swalla, 2008). Hemocytes within the stolon of connected zooids appear to be significant in terms of budding as they share characteristics of progenitor stem cells in some species, allowing them to form the epithelial tissues required in a new individual during vascular budding (Gutierrez & Brown, 2017; Hirose & Akahori, 2004; Oka & Watanabe, 1957; Tiozzo et al., 2008; Brown & Swalla, 2008). Gutierrez and Brown (2017) found two new blood cells with progenitor stem cell-like features within the compound colonial ascidian Symplegma brakenhielmi. Brown and Swalla (2008) also highlight the role of stem cells within the hemocyte of Botrylloides violaceus. Although not socially colonial ascidians, their research shows the diversity and plasticity in budding for reproductive or regenerative purposes amongst the Ascidiaecea.

Perez-Portela et al. (2009) suggested classifying Stolidobranchia based on vascular systems rather than through a distinction between solitary and colonial ascidians. They found that the paraphyletic nature of the Styelidae hints at the origin of coloniality occurring individually multiple times within the family, including aggregations of social ascidians connected via stolons (Ritter 1897, Perez-Portela 2009). From this, they suggest that ascidians should be considered in terms of the presence of a linking vascular system between aggregated colonies, and those colonies without a common system. It is proposed that viviparity may have been a required adaptation leading to coloniality (Alie et al. 2018, Perez-Portela et al. 2009), as non-embryonic development often involves budding of a fully developed zooid after brooding, much like viviparous development (Zeng et al. 2006).

Figure 5

Biogeographic Distribution

Colonial ascidians are found worldwide in tropical and temperate oceans (Abdul et al. 2016). Their variation in budding methods allows species of all three sub-orders to be highly invasive. Species that reproduce sexually as well as asexually have a high dispersal as larvae can travel on currents. Research into the regenerative properties of stolons suggest they can disperse through broken stolons or zooids that have been separated from the colony (Deviney, 1934).

Stolons can occur as vertical stems connecting individuals like grapes on a vine. Others are flat venous tissue that may potentially adhere to rocky and sandy surfaces. A stolon allows the ascidians to exist within a wide distribution: rocky shores, intertidal zones, reefs and benthic communities (Buss et al. 1979). Due to their successful colonising technique, some social ascidians are widely invasive as they biofoul onto ships. When the stolon is broken, individual zooids can regenerate through budding from the vasculature and this gives them an advantage in colonising new areas and dispersal. Species with long stolons have been shown to be able to grow over bryozoans, algae and larger solitary ascidians which gives them a competitive advantage when settling in benthic communities (Hughes, 1989).

The specimen observed on the ARMS plates (refer to Fig 1 and 2.) was found on the bottom of the plate, facing the sea floor in Dunwich Bay: subtropical waters with boat traffic.

Evolution and Systematics

Colonial and solitary ascidians are found in Stolidobranchia (with only Styelidae having colonial species) and Phlebobranchia, whereas Aplousobranchia is entirely colonial (refer to Figure N; Monniot et al. 1991).

As ascidians are soft bodied, there is a lack of fossil evidence that can be used to identify the evolutionary pathway of coloniality and solitary (Brown & Swalla, 2008). Often, the morphological and phylogenetic analyses aren’t consistent with one another, which makes it difficult to draw conclusions about the convergence or homology of the trait. However, Zeng et al. (2006) suggest that colonialism evolved once within the Stolidobranchs from a solitary ancestor, as they found the phylogenetic information aligns with the morphological evidence (Fig. 6).

Molecular data has shown that coloniality has evolved once within the Order Stolidobranchia. Stolidobranchia is a monophyletic clade, and research of mitochondrial and nucleic DNA has shown Pyuridae were mostly monophyletic, while Styelidae contains many clades (Ritter, 1897; Perez-Portela, 2009). It is proposed that viviparity may have been a required adaptation leading to coloniality (Alié et al., 2018; Pérez-Portela et al., 2009), as non-embryonic development often involves budding of a fully developed zooid after brooding, much like viviparous development (Zeng et al., 2006). Solitary and colonial species are found across the Ascidiaecea phylogeny suggesting that coloniality has either been gained or lost independently multiple times (Brown & Swalla, 2012). Brown and Swalla (2012) have shown that different budding mechanisms are found within the colonial specimens of the three ascidian orders: Stolidobranchia, Phlebobranchia and Aplousobranchia which supports that coloniality is a convergent trait.

The Stolon Hypothesis

The tail of tunicates was often thought to be the structure related to the trunk in more advanced chordates, however salps and pyrosomes are members of Tunicata but have lost the presence of a tail in the larval stage and in it’s place evolved a reproductive stolon (Lacalli 1999). Lacalli (1999) proposed that salps and pyrosomes would have to be ancestral to Ascidiaecea for this to be true. Instead, he describes the ‘stolon hypothesis’: the idea that from a stolon similar to those of salps (lined with blastozooids) evolved the trunk and tail in chordates. The proposal of this hypothesis emphasises the relationship of Tunicata (and hence ascidians) to chordates. The stolon in colonial ascidians is also used in reproduction, and the presence of a tail in the larval stage suggests that Ascidiaecea are more closely related to more advanced chordates (refer to Figure 5.). Overall, Lacalli’s research highlights the importance of asexual reproduction to the evolutionary history of the chordates.
Figure 6

Conservation and Threats

The wide distribution of social ascidians and their plasticity in morphology and budding means that they are not threatened. The stolon of some colonial ascidians has adhesive properties that allows them to biofoul on large ships and jetty’s. Proteins within the stolon allow it to adhere to surfaces, withstanding currents and other disturbances, which can lead to highly invasive species (Li et al., 2019). The spatially competitive advantage that stolonic budding offers colonial over solitary ascidians also lends itself to the invasiveness of species (Buss, 1979). As such, the non-embryonic development and stolons of social ascidians lend themselves to a persistent lifestyle through high colonisation, rapid growth and regeneration.


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