Trying to identify an invasive colonial Sea Squirt from the Gulf of California.

Invasive species are one of the greatest challenges faced by conservationists today, often rapidly taking over ecosystems where they have no natural enemies and displacing endangered or commercially important local species in the process. The first step in controlling an invasive species is identifying it, which enables appropriate control systems to be introduced. However, this is often difficult and complicated, as invasive species are not necessarily well understood, or even known, in their own ecosystems. This is particularly true in marine environments, where our taxonomic understanding of many groups is poor, and increasingly plagued by a lack of skills as few young scientists are attracted into the field. This is despite the problem of invasive species being particularly acute in marine environments, where international shipping traffic has made it easy for many benthic 'fouling' organisms to rapidly establish global distributions.

Ascidians, or Sea Squirts, are the most abundant class of the Subphylum Tunicata and are distributed along shorelines worldwide. They are sessile marine invertebrates and are widely used as a model organism for developmental and evolutionary studies. Ascidians exhibit multiple morphological characteristics, from small colonial to colorful and large solitary forms. They are divided into three major well-accepted orders, namely, Phlebobranchia, Aplousobranchia, and Stolidobranchia, based on the branchial sac morphology of the adults. However, the class Ascidiacea is paraphyletic (i.e. not everything thought to be descended from the last common ancestor of the group is considered to be an Ascidian) with the  Phlebobranchia and Aplousobranchia showing a close relationship with Thaliaceae (Pyrosomes, Salps, and Doliolids), a non-Ascidian Tunicate class, whereas the Stolidobranchia remains a distinct and monophyletic group. Over the course of several decades, the Ascidiacea have been shown to be an important class of ecological species because of their invasive potential along with their ability to adapt to new environments. Transportation of Ascidians attached to ship hulls as fouling material and within the ballast water of ships has enabled them to invade many new territories. This phenomenon has major impacts on local marine biodiversity as well as aquaculture industries. Therefore, the Ascidiacea are now considered as important model species for the study of non-indigenous species worldwide.

Sea Squirts are particularly problematic from a taxonomic point of view, with a simple body plan which provides few diagnostic features, and very few scientists specializing in their taxonomy. To make matters worse, the group appears to be rife with cryptic species (i.e. species which are physically identical or nearly identical, but biologically distinct), so that many identified species are likely to be clusters of similar-looking species, often with different ecological constraints. Many species of Sea Squirt have proven to be adept at colonizing new environments, where they are often identified as new species, so that particularly successful invasive species often have several different names, further adding to the taxonomic confusion within the group. A combination of careful physical examination and genetic analysis can hopefully unravel some of these problems, particularly in the identifying of pseudo-indigenous species, i.e. invasive species thought to be native to their new habitat because their origin is unknown.

Eleven invasive species of Sea Squirt had been identified from Mexican waters by 2014, five of them from the Gulf of California. In 2015, a colonial Sea Squirt previously unknown to the region was identified in Ensenada de La Paz, which spread rapidly, causing a mass mortality event in Pen Shells, Atrina maura, a Bivalve species commercially important locally, whose large shells provided an ideal substrate for the invaders. The Pen Shell fishery was already suffering from over-harvesting, with a moratorium on their collection introduced in 2013 to allow the population to recover, something which appears to have been significantly hampered by the arrival of the Ascidians (no significant variation in other environmental variables was detected which coincided with the die-back). A subsequent biofouling experiment carried out in Bahía de La Paz found that the Sea Squirts, tentatively identified as Distaplia cf. stylifera, were the most abundant macro-organisms settling on a series of silicone resin coated metal panels placed in the water for two months. Furthermore, the Sea Squirt colonies provided a substrate upon which 28 species of epibiont Polychaete Worms were able to settle, further adding to the biofouling. Subsequent studies of the Sea Squirts, which have continued to suppress Shellfish populations, and become a serious biofouling problem in the area, have used the name Distaplia stylifera, although this is only an assumption, based upon a rough similarity to descriptions and the fact that it is a common invasive species. No formal taxonomic identification of the invader has been attempted to date, something which may be hampering efforts to find an effective control.

In a paper published in the journal ZooKeys on 5 April 2023, Betzabé Moreno-Dávila and Leonardo Huato-Soberanis of the Programa de Ecología Pesquera at the Centro de Investigaciones Biológicas del Noroeste, Jaime Gómez-Gutiérrez of the Departamento de Plancton y Ecología Marina at the Instituto Politécnico Nacional, Carolina Galván-Tirado of the Consejo Nacional de Ciencia y Tecnología, Carlos Sánchez of the Departamento de Ciencias Marinas y Costeras at the Universidad Autónoma de Baja California, Teresa Alcoverro of the Department of Marine Ecology at the Centre for Advanced Studies of Blanes, Eduardo Balart, also of the Programa de Ecología Pesquera at the Centro de Investigaciones Biológicas del Noroeste, and Xavier Turon, also of the Department of Marine Ecology at the Centre for Advanced Studies of Blanes, present the results of a study of the invasive Sea Squirt from Ensenada de La Paz, which examines the Mexican species' morphology and compares it to previous descriptions from other parts of the world, as well as attempting a genetic analysis.

Bahía de La Paz is located on the southeastern tip of the Baja California Peninsula. The Ensenada de La Paz is separated from Bahía de La Paz by a 12 km long sandbar known as El Mogote. The Ensenada de La Paz has an area of about 45 km² and a maximum depth of 10 km and a bottom covered by patches of sand and mud-silt. The Ensenada de La Paz and the Bahía de La Paz are connected by a shallow channel about 1 km wide and 4 km long, upon which are seven yacht docks associated with the city of La Paz, while at the entrance lies the Pichilingue commercial harbour.

(A) Area of study in Ensenada de La Paz located in the southern part of Bahía La Paz, Baja California Sur, Mexico. (B) Both bodies of water are located on the southwest coast of the Gulf of California, Mexico. (C) Sampling sites and potential sources of dispersal of Tunicates (circles). Moreno-Dávila et al. (2023).

Colonies representing three different colour-morphs of the invasive Sea Squirt (white, orange, and purple) were collected from four sites within the Ensenada de La Paz, for a total of twelve colonies, which were preserved for morphological analysis as well as having their DNA extracted for genetic analysis. The colonies were found growing at depths of 0.5-3 m, growing on the shells of Sea Pens, a PVC pipe, buoys, a rope, and wooden docks.

'Distaplia stylifera' from Ensenada de La Paz. (A) Live orange colony. (B) Live purple colony. (C), (D) Typical mushroom-shaped colonies. (E) Zooid. Scales bars: 10 mm (A), (B); 2.5 mm (C), (D); 1 mm (E). Moreno-Dávila et al. (2023).

The colonies were predominantly orange, and approximately mushroom-shaped, with mottled white markings around the common cloacal-aperture, with purple and white colonies also present within the population. They reach a maximum of about 2 cm, with the head having a diameter of up to 2.5 cm. The colonies are more heavily pigmented around their tips than at the base, with pigment only remaining in this area after preservation. The tunics of the colonies are firm, and the stalks sometimes branch, so that two or more colonies share a common base; some colonies lack stalks altogether, forming cushion-shaped masses that spread over the substrate. The head of each colony is shared by one or more zooid systems, each sharing a common cloacal-aperture, which is surrounded by a single or double ring of zooids.

The zooids are up to 5 mm in length, excluding the gonadal sacs. Each zooid is divided into a thorax and abdomen. Two sacs are attached to this structure, a smaller one, containing the gonads, is attached to the right posterior side of the abdomen. The second sac contains embryos and developing larvae, and is often longer than the zooid. This second sac is attached to the posterior part of the pharynx by a thin peduncle.

'Distaplia stylifera'. (A) Zooid (thorax and abdomen). (B) Thorax. (C) Dissected thorax. (D), (E) Stomach. (F) Gonads. (G) Larvae. (H) Enlargement of one larva showing two pigmented spots. Scale bars: 10 mm (A); 0.5 mm (B), (C), (F), (G); 0.25 mm (D), (E). All images except (F) correspond to stained zooids. Moreno-Dávila et al. (2023).

The oral siphon on the thorax is smooth-rimmed or has six slight lobulations, and a large atrial opening which exposes most of the branchial sac. A  wide flap-like lid with smooth or lobed margins called the atrial languet lies on top of the atrial opening. This languet is crossed by several transverse muscular bands. Each side of the thorax has about 30 bands of longitudinal muscle. About 14 simple oral tentacles are also present on the thorax. The pharynx has four stigmata rows clearly divided by parastigmatic vessels, with the first two rows typically having 18-19 stigmata, and the posterior two rows having 15-16. Three simple dorsal languets are found between these rows, slightly displaced to the left.

'Distaplia stylifera' (A) Zooid (thorax and abdomen). (B) Abdomen. (C) Larva. Abbreviatures: a. anus; am. ampullae; ap. adhesive papillae; oc. ocellus; o. oocyte; pv. parastigmatic vessels; pg. pyloric gland vesicle; sc. statocyte; sg. stigmata; st. stomach; t. testes. Scales bars: 1 cm (A); 0.5 mm (B), (C). Moreno-Dávila et al. (2023).

Within the abdomen lies an elongated and curved stomach, the wall of which has more than 20 fine longitudinal plications (folds), which can be seen on the inner and outer surfaces when the specimens are sectioned, although they are sometimes interrupted or divided. A short post-stomach connects to an enlarged mid-intestine at the bottom of the gut-loop. The distal intestine runs to the anterior, and ends in a bilobed anus at the base of the atrial aperture. A pyloric (mucus) gland between the stomach and the intestine and continues anteriorly forming sinuous tubules over the intestine in front of the stomach.

The gonads are held within a pedunculated sac, with one or two oocytes at the bottom, above which lie a cluster of five or six elongated or wedge-shaped testes. A  common sperm duct arises posteriorly from the cluster of testes, but turns anteriorly at its very beginning, without overlapping the oocytes.

All of the colonies examined had larvae incubating in long sacs that reach posteriorly deeper than the zooids themselves in the colonies. These sacs typically contained one or two well developed larvae, about 1.3 mm in length, plus three embryos. When fully developed the larvae reach about 1.5 mm, and possess three adhesive papillae, two dorsal and one ventral, with a globular ampulla each in the stalks. The four rows of stigmata have developed by this stage, with an incipient abdomen folded under the branchial sac. A sensory vesicle contains two pigmented spots, with a larger one above and a smaller one below, though these can be hard to differentiate, as they are close together and the larvae are not transparent. 

Distaplia stylifera was originally described in the Red Sea, and subsequently has been recorded from several areas of the Indo-Pacific, Australia, the Philippines, and the South China Sea, as well as. under the possible synonym Distaplia mikropnoa, from Palau. There have been reports from the Mediterranean, although these are considered dubious. It has also been reported in the Western Atlantic, from the coast of North Carolina south as far as Jamaica, and at several locations in the Caribbean, and possible further south around Sao Paulo in Brazil. It has not, however, previously been reported from the Eastern Pacific.

Sites of previous records of Distaplia stylifera: (1) Red Sea, 1874 (type locality). Indo-Pacific; (2)-(9). Mediterranean, (10). Western Atlantic Ocean, (11)-(18). Eastern Pacific Ocean, (19) present study. The type locality in the Red Sea and the record of the present study are indicated with stars. Moreno-Dávila et al. (2023).

Despite successful amplification of DNA apparently collected from the Ascidian colonies, all of this proved, upon analysis, to be closer to that of Algae, Bacteria, or Fungi, than to any Animal, despite great care being taken to avoid any contamination. For this reason the taxonomic comparison had to be made entirely upon physical examination of the specimens, and comparison to other members of the genus Distaplia.

The taxonomy of the genus Distaplia is mainly based on characters such as colony shape, arrangement of zooids in systems, presence or not of gonadal sac, stigmata per row, stomach shape and external surface, and muscle arrangement. Based upon these criteria, the Ensenada de La Paz specimens would appear to be entirely consistent with Distaplia stylifera. However, Moreno-Dávila et al. note that the original description of the species was made in 1874, and like a lot of descriptions from this period, was a lot less detailed than descriptions made by modern taxonomists. Furthermore, that description was made upon a specimen that was probably a juvenile, lacking larvae and having under-developed gonads. 

Descriptions of populations of modern populations of 'Distaplia stylifera' show some variation, making it possible that the global population is in fact a cluster of closely related species. Notably, the specimens collected at Ensenada de La Paz all had parastigmatic vessels, something also reported in almost all specimens collected from Australia, as well as specimens collected from the Caribbean. An absence of such vessels has been noted in specimens from Madagascar, and it has been suggested that the presence or absence of these vessels might be a feature which can be used to differentiate between Distaplia stylifera and Distaplia mikropnoa. However, a number of other features can be used to differentiate Distaplia mikropnoa, which include a long double rows of zooids converging to the terminal common cloacal apertures, a long post-pyloric part of the gut loop, and a lack of a gastric reservoir. Furthermore, in Distaplia mikropnoa the course of the gastro-intestinal ducts that does not cross from the stomach to the ascending limb of the gut loop but extends down the descending loop. Based upon this, Patricia Kott concluded that the two species are both valid, but that they have widely confused within the literature. Notably, she concluded that the Palau population belonged to Distaplia stylifera rather than Distaplia mikropnoa.

Oval follicles have been suggested as another feature which could be used to differentiate the two species, with specimens which have up to 15 oval follicles assigned to Distaplia mikropnoa, while those having only five or six, which would include the Ensenada de La Paz population, belong to Distaplia stylifera. 

The sperm duct is another feature apparently variable in populations assigned to Distaplia stylifera, with some populations having a sperm duct running posteriorly and making one or several loops over the oocytes before turning anteriorly, while in other populations, including the Ensenada de La Paz population, it is straight. Furthermore, in some populations, including Ensenada de La Paz, the gonadal sac is attached via a peduncle, while in others it is almost flush with the abdomen, separated by a wide neck. 

Moreno-Dávila et al. suggest that this wide range of variable features indicate that 'Distaplia stylifera' is probably a cluster of closely related species, often mistaken with the closely related Distaplia mikropnoa. This probably cannot be resolved without a more detailed taxonomic study, using both morphological and genetic methods to analyse populations from different regions of the world. Nevertheless, the reporting of a member of the species cluster from the Eastern Pacific represents a significant range-expansion for a group previously known from tropical regions of the Western Atlantic, Indian Ocean, and Western Pacific. 

Moreno-Dávila et al. were unable to extract DNA from their specimens of 'Distaplia stylifera', and note that no records exist within the GenBank and BOLD public databases. The co-generic Distaplia bermudensis is present in these databases, and shows a 14–20% genetic variance between different populations and morphotypes, indicating either a remarkable level of genetic variation within a single species, or that Distaplia bermudensis is also a species cluster. 

This taxonomic uncertainty makes it difficult to assess to what extent the different populations of 'Distaplia stylifera' represent introductions or local species, although Moreno-Dávila et al. note that the populations from North Carolina and the Caribbean appear to strongly favour lagoons and artificial structures, which is behaviour typical of invasive fouling organisms, and that this population appears to be expanding southwards to the coast of Brazil, where again it has been found only on artificial substrates. 

Despite this uncertainty, Moreno-Dávila et al. believe that the Ensenada de La Paz population does represent a high-impact invasive species, and given the absence of any other known populations of 'Distaplia stylifera' in the Eastern Pacific, and the behavioural similarities of this species to the Western Atlantic populations, one which has probably arrived via the Panama Canal. probably crossing the canal on a large ship, then secondarily transferring to a smaller vessel capable of entering the harbours around La Paz. 

See also...

Follow Sciency Thoughts on Facebook.

Follow Sciency Thoughts on Twitter.

Biomineralising Tunicates from the Late Triassic of Italy.

Biologically controlled biomineralisation is a fundamental process in the evolution of the animal kingdom. Though it was sporadically achieved already in the latest Precambrian, the so-called 'Cambrian Explosion' around 540 million years ago, marks the general advent of Animals with hard parts in the fossil record. It is well known, that this event was not an instantaneous process, but that it was accomplished in a time span of about 20–25 million years. The acquisition and evolution of hard skeletons within the great majority of phyla and classes are the basic prerequisites for the reconstruction of animal form and function through time. Once hard parts were created in order to protect and stabilize soft tissues and organs, this evolutionary advantage indeed developed a great variety of morphologies, but in spite of this diversity and modification, solid skeletons were generally maintained as such until extinction of the equivalent groups.

In a paper published in the Journal of Paleontology on 24 January 2020, Jobst Wendt of the Fachbereich Geowissenschaften der Universität Tübingen, describes and decrypts an unusual case of biomineralisation in fossils of the rather poorly known class Ascidiacea (Sea Squirts) of the subphylum Tunicata.

The reduction of a hard skeleton into a mere softbodied stage seems to have been achieved in some Coleoid Cephalopods and Opisthobranch Sea Slugs, but this accordance is based on notional palaeontological evidence. An alleged late acquisition of a hard skeleton inherited from soft-bodied ancestors is suspected in Bryozoans and Scleractinian Corals, but in contrast to Ascidians, once these groups had acquired calcified skeletons, they maintained this protective and stabilizing advantage until Recent. Thus, the very late acquirement of a solid calcareous skeleton among Tunicates and its later loss in favor of an almost soft-bodied stage is a very rare phenomenon in the geological record.

From their form and mode of life, Ascidiacea (Sea Squirts) appear as rather simple-structured organisms that superficially and functionally are evocative of Porifera. But the presence of a tubular dorsal nerve cord and a notochord in their larval stage and pharyngeal clefts allocates them amuch higher position within the Animal Kingdom as a subphylum of the Chordata. Ascidians are an artificial, polyphyletic group that comprises three orders (Aplousobranchiata, Stolidobranchiata, Phlebobranchiata) with a total of about 2940 living species. They generally lack hard parts, thus minimizing the possibility of preservation. Only a few genera of the orders Aplousobranchiata and Stolidobranchiata segregate tiny isolated spicules, embedded in the mantle (tunica) or other organs, but their origin remains unknown. Tunica is the Latin word for mantle. Therefore, many zoologists regard both terms as synonymous. Others, however, distinguish an inner layer (tunic), in which the spicules are secreted, from an outer one (mantle) In order to guard against misunderstandings, Wendt uses both terms as synonyms because, with regard to fossils, the differential application of both terms would appear arbitrary.

Spicules of cpossible Tunicates have been found in rocks as old as lower Liassic. Generally, ascidian spicules consist of aragonite, more rarely of vaterite or other minerals. Unlike some siliceous sponge spicules, they are never fused together and thus could not form solid skeletons. The discovery of complete soft-bodied ascidians (lacking spicules, however) in the lower Cambrian of China has revealed that ascidians existed already at an early stage of Metazoan evolution, but this finding does not allow to reconstruct their relationships to Mesozoic to recent representatives of this class. A new evolutionary branch among Ascidians is the recent discovery of calcareous Tunicate exoskeletons in the Upper Triassic and their ancestors in the Permian. 

Wendt suggests that these very unusual skeletons, which are composed of irregular aragonitic plates, can be assigned to no phylum other than the Tunicates. This systematic position of these previously unknown organisms to the class Ascidiacea has been the fundamental clue for the systematic attribution of the new discoveries described by Wendt. The fact, however, that they are endoskeletons, opens an unusual insight into a new aspect of the evolution of this poorly known class which has no counterpart among their living representatives. Tunicate exoskeletons have long been known from Permian deposits in east Asia and Europe, but until recently they were erroneously attributed to Rugose Corals. These skeletons are composed of a varying number (2 to about 35) of irregular plates that consist of acicular aragonite. This very unusual construction and mineralogy were crucial for their systematic attribution, though similar living representatives of this class are unknown. Comparably organized endoskeletons now add a new aspect to the fossil record of this largely ignored subphylum of the Chordata.

Endoskeletons are widespread in the Animal Kingdom and display a great variety of shape, function, and mineralogical composition. They are typical for the major phyla of Deuterostomia (Echinodermata and Vertebrata, including Conodontophorida) and many Protista (Foraminifera and Radiolaria), which exhibit a stunning variety of shapes and geometries. Endoskeletal spicules (sclerites) of different mineralogical composition evolved independently among several phyla and classes (Porifera, Octocorallia, Vermes, Holothuroidea, Tunicata). Apart from the calcareous octocoral Tubipora musica, only some Porifera only some Porifera (Hexactinellida and Lithistida), produced siliceous spicules, which are fused or articulated into compound endoskeletons forming cubic or irregular meshworks, thus giving more stability to the soft body. Among other phyla, compact solid calcareous endoskeletons occur only among the Mollusca. Composite calcareous endoskeletons consisting of numerous plates with flexible boundaries are characteristic for Echinodermata and for the newly discovered fossil Tunicata.

Wendt describes two incomplete specimens from the the Cassian Formation (lower Carnian) of the Dolomites (northern Italy). Extensive searches for additional, either biostratigraphically older, contemporaneous, or younger specimens from European and North American reef specialists and collections were unsuccessful. The two specimens were discovered among far over a million of skeletal remains from the the Cassian Formation. The number of taxa and the diversity of skeletal remains collected from the Cassian Formation during almost two centuries are indeed impressive and assign this rock unit a singular rank among the Fossil-Lagerstätten in the geological record. As of 2019, 1429 species have been described so far from this formation. A special feature of this unrivaled fauna is their often excellent state of preservation, exemplified by diagenetically almost unaltered aragonitic microstructures, which are among the oldest in earth history. In this state of perfection and considering its age, the Cassian Fauna can even be regarded as unique.

Fossil record of Tunicates. Black dot, soft-bodied; black triangles, calcareous exoskeletons; black square, calcareous endoskeletons; asterisks, spicules. Wendt (2020).

The fossils are placed in a new order and family of Ascidians, named the Cassianosomidae and Cassianomorpha respectively; both names refer to the Cassian Formation. Both are placed in the new genus Toscanisoma, which is named in honour of Maria Luigia Toscani, who collected the fossils upon which the study is based. Each of the two fossils is described as a separate species within the new genus.

The first species described is named Toscanisoma multipartitum, where 'multipartitum' means 'consisting of several parts'. This is a sessile colonial species consisting of seven branching tubes (zooids) composed of irregular plates that are joined by straight or curved boundaries on the outer and zigzag ones on the inner side. The only complete zooid of the colony is closed at the top by six plates that are smaller than the remaining ones. The other zooids are incomplete, but were probably slightly larger. Spicules have not been observed.

Toscanisoma multipartitum holotype (GPIT/TU 82). (1) Lateral view (drawing); (2) opposite side; (3) view from top; (4) base. Asterisks mark complete zooids, arrows indicate zigzag sutures on inner surfaces. Scale bar 10 mm. Wendt (2020).

The only specimen of Toscanisoma multipartitum available is not complete, lacking the basal portion, which probably served as a holdfast. The seven zooids, which bud from the flattened base, have different shapes ranging from almost circular to elongate or flattened in cross section. Only the smallest zooid is complete, 25 mm long and consists of about 10 plates with very faint and barely recognisable outer boundaries. The inner plate boundaries of the incomplete zooids show zigzag sutures similar to contemporaneous representatives of the order Khmeriamorpha. The zooids fit closely together at the base and are separated by small open interspaces higher up.

The second new species is named Toscanisoma triplicatum, meaning 'triplicate'. as the only known specimen consists of three zooids. This species is distinguished from Toscanisoma multipartitum by the presence of spicules, the different kind of budding, in which the separation into three individual zooids starts at a distance of about 2 cm above the base. Thus, in their early growth stage, the individual zooids share the outer walls of the adjacent ones, but higher up than in Toscanisoma multipartitum.

Toscanisoma triplicatum holotype (GPIT/TU 83). (3) Lateral view; (4) view from base; (5) view from top; 1, 2, 3 mark cavities of three incomplete zooids. Scale bar 15 mm. Wendt (2020).

Because of the fragileness of the specimen, the inner side of the basal cup and the individual zooids could not be sufficiently prepared, but it is assumed that the inner plate boundaries show zigzag sutures similar to Toscanisoma multipartitum. Due to the incomplete state of preservation and the lack of comparable material, speculations about the final growth form and the number of zooids are premature.

The morphological and mineralogical features described by Wendt raise the pivotal question of the systematic attribution and the functional morphology of these enigmatic remains. Solid skeletons (whether exo- or endo-) composed of irregular plates that consist of acicular aragonite crystals are unknown in the Animal Kingdom, with one exception: Permian/Triassic Ascidian Tunicates with a compound calcareous skeleton, which appeared in the early(?) Permian and became extinct during the Late Triassic. These remains reveal a certain relationship to some living Ascidians (e.g. Chelyosoma and Forbesella), although these lack any hard parts. But they are partially composed of irregular soft plates that can be moved by muscles. The hinge-like sutures on the inner plate surfaces of the newly described fossil endoskeletons also suggest a certain flexibility of the compound skeleton, which is indispensable for their here proposed assignment to Ascidian Tunicates. The latter are filter-feeders in which a steady flow of seawater moves through an atrial and a branchial siphon. If we assign a similar function to the fossil counterparts described by Wendt, it must be required that one or two of the top plates could be opened by muscles during the life-time of the organism because this is the case in the genera Chelyosoma and Forbesella. Unfortunately, the upper part of the examined specimens generally is not preserved. But one zooid of Toscanisoma multipartitum is complete and exhibits a mosaic of tiny plates, which probably could be opened by muscles to allowaccess for the protruding siphons.

At a first glance, the presence of a solid calcareous endoskeleton in Cassianomorpha might be surprising. However, one should bear in mind that, apart from Arthropoda (which have a totally different kind of growth), all invertebrate classes with a calcareous exoskeleton show well-developed growth lines reflecting an intermittent growth at the edge of the skeleton-secreting tissue. This is not the case in the Cassianomorpha in which the skeleton is formed within the mantle (tunica) starting more or less simultaneously at several nucleation points. Faced with these observations, it is less surprising that the extinct Cassianomorpha developed an endoskeleton, as did the other main Deuterostome phyla or classes (Echinodermata, Vertebrata, Jurassic to recent Ascidiacea), than the fact that the other contemporaneous fossil Ascidian order Khmeriamorpha strangely developed an exoskeleton.

By mid-Cambrian times, representatives of almost all Metazoan phyla and subphyla had reached a high degree of biomineralisation in such away to enable a reliable reconstruction of their evolution. Why is this not the case with Tunicates, which must have existed contemporaneously? It is possible to speculate about a change in seawater chemistry or a possible global perturbation of the carbon cycle near the Carboniferous/Permian boundary, but such speculations about the appearance of mineralised (aragonitic) tunicate skeletons at this interval appear rather theoretical, because the time-span of an 'aragonitic ocean' (Mississippian to Middle Jurassic) is not consistent with the existence of aragonitic Ascician skeletons.

A possible answer to this fundamental question is that compound pre-Permian tunicate skeletons might have existed in earlier Paleozoic times, but they have not yet been discovered or recognized as such. It can also not be totally dismissed that they are hidden among the great number of previously described fossil Problematica or those of incertae sedis. The discovery of rare spicules embedded in the solid skeleton clearly points to a new and successful attempt of Tunicate biomineralisation in the Late Triassic, which persisted until recent. However, post-Triassic survivors of Tunicates with compound calcareous, either endo- or exoskeletons, are unknown from the fossil record. Wendt speculates that post-Triassic Ascidians developed other protective strategies that served as a defensive function (e.g., secretion of indigestible chemicals), which, of course, cannot be detected in fossil remains. Not taking into account these theoretical considerations, the total loss of a compound calcareous skeleton in Ascidians during the Late Triassic in favor of a much less stable one consisting of isolated spicules only remains an unresolved question.

See also...

Follow Sciency Thoughts on Facebook.

Investigating the genetic diversity of Sea Squirts in Rongcheng Bay, Shandong, China.

Ascidians, or Sea Squirts, are the most abundant class of the subphylum Tunicata and are distributed along shorelines worldwide. They are sessile marine invertebrates and are widely used as a model organism for developmental and evolutionary studies. Ascidians exhibit multiple morphological characteristics, from small colonial to colorful and large solitary forms. They are divided into three major well-accepted orders, namely, Phlebobranchia, Aplousobranchia, and Stolidobranchia, based on the branchial sac morphology of the adults. However, the class Ascidiacea is paraphyletic (i.e. not everything thought to be decended from the last common ancester of the group is considered to be am Ascidian) with the  Phlebobranchia and Aplousobranchia showing a close relationship with Thaliaceae (Pyrosomes, Salps, and Doliolids), a non-Ascidian Tunicate class, whereas the Stolidobranchia remains a distinct and monophyletic group. Over the course of several decades, the Ascidiacea have been shown to be an important class of ecological species because of their invasive potential along with their ability to adapt to new environments. Transportation of Ascidians attached to ship hulls as fouling material and within the ballast water of ships has enabled them to invade new territories. This phenomenon has major impacts on local marine biodiversity as well as aquaculture industries. Therefore, the Ascidiacea were recently considered as important model species for the study of nonindigenous species worldwide.

In a paper published in the journal Ecology and Evolution on 10 March 2020, Punit Bhattachan and Runyu Qiao of the Key Laboratory of Marine Genetics and Breeding at the Ocean University of China, and Bo Dong, also of the Key Laboratory of Marine Genetics and Breeding at the Ocean University of China, and of the Laboratory for Marine Biology and Biotechnology at the Qingdao National Laboratory for Marine Science and Technology, and the Institute of Evolution and Marine Biodiversity at the Ocean University of China, present the results of a comparative analysis of three Ascidian species from northeast of China, with samples from elsewhere in the world, using the cox1 gene sequence as a genetic marker to distinguish native from invasive ascidian populations.

Bhattachan et al. collected adults of three Ascidian species, Ciona robusta, Ciona savignyi, and Styela clava, from the Rongcheng Bay area of Shandong Province, which is a part of the Yellow Sea, in northeast China. These were maintained in the laboratory in seawater tanks with aeration and constant illumination, where species were identified morphologically, and internal tissues were collected for DNA extraction and sequencing.

Collection site of the Ascidian samples (black arrow). Bhattachan et al. (2020).

The cox1 sequences from three ascidian populations at different regions of the world were retrieved from the NCBI database to build multiple sequence alignments. Only the unique haplotype datasets were used for the multiple sequence alignments. Neighbor-Joining  and maximum parsimony  methods were employed to construct a phylogenetic tree with 1000 bootstrap estimations in the default setting using MEGA7.0. The barcode region of the cox1 sequence (accession no. HM151268.1) of the Sea Pinapple, Halocynthia roretzi, was used as an out-group.

Multiple sequence alignments of cox1 from the three Ascidian species were performed separately in ClustalW hosted by MEGA7.0 using default settings. Genetic diversity parameters, including haplotype number, haplotype diversity, nucleotide difference, mutation number per sequence, number of segregating sites, and nucleotide diversity, were estimated using DnaSP software.

Relationships among the three Ascidians cox1 haplotypes found globally, including those from China, were determined using a median-joining method in the network software. To infer the population structure and understand the connectivity between native and invasive ascidian populations, we performed molecular variance analysis using cox1 haplotypes from samples available in the database as well as those in Bhattachan et al.'s dataset study using the ARLEQUIN 3.11 software.

Morphological identification of the three Ascidian species. (b) Ciona robusta adult with oral siphon (os), atrial siphon (as), sperm duct (white arrow), oviduct (black arrow), and red colour at the tip of the sperm duct (arrowhead). (c) Ciona savignyi adult with oral siphon (os), atrial siphon (as), sperm duct (white arrow), and oviduct (black arrow). (d) Adult Styela clava with oral siphon (os) and atrial siphon (as). Scale bar represents 1 cm. Bhattachan et al. (2020).

Bhattachan et al. cloned the full length of the cox1 gene from genomic DNA of 50 individuals of the three Ascidian species. Each resulting sequence was subjected to BLASTN, with the results indicating that these sequences belonged to the three respective ascidian species. The open reading frames of the cox1 sequence from three species were variable. Bhattachan et al. identified a deletion polymorphism of cox1 in Ciona savignyi, but not in Ciona robusta and Styela clava. For instance, only a single 1560 and 1543 base pair-length of cox1 sequence was identified in Ciona robusta and Styela clava, respectively, whereas two different lengths of cox1 (1545 and 1548 base pairs) were identified in Ciona savignyi. All these sequences were deposited in the NCBI database.

Bhattachan et al. also retrieved the cox1 barcode sequences from the NCBI database Only the cox1 barcode regions of unique haplotypes were used for multiple sequence alignments and phylogenetic tree construction. The resulting phylogenetic trees allowed us to delineate different haplotypes among all of the samples. In the Ciona robusta tree, Bhattachan et al. found that the haplotypes (H_1 to H_9) from China did not form a single clade in either Neighbor-Joining or maximum parsimony trees, but rather clustered with some haplotypes from individuals originating from Korea and the USA. Similarly, the haplotypes (H_1 to H_16) of Ciona savignyi from China did not cluster in a single clade in either Neighbor-Joining or maximum parsimony trees. Instead, they grouped with other haplotypes from Korea and the USA. In addition, Neighbor-Joining and maximum parsimony trees did not resolve the haplotypes (H_1 to H_14) of Styela clava from China into a single clade either. Conversely, they formed a cluster with some haplotypes from New Zealand and the USA, which were invasive populations.

Bhattachan et al. used the cox1 gene for molecular diversity analysis. Nine haplotypes were identified among 14 Ciona robusta samples, 14 haplotypes among 19 Styela clava samples, and 16 haplotypes among 17 Ciona savignyi samples. The results of the comparative analysis using different genetic diversity parameters also revealed that Ciona savignyi was diverse compared with Ciona robusta and Styela clava. The haplotype diversity was comparatively higher in Ciona savignyi (0.993 + 0.038) than that in Ciona robusta (0.912 + 0.059), and Styela clava (0.947 + 0.038). Similarly, the detected average number of nucleotide difference in Ciona savignyi (20.618) was higher than that in Ciona robusta (8.143) and Styela clava (11.550). Nucleotide diversity and average number of mutations were also relatively higher in Ciona savignyi (0.02630, 0.05061) compared with Ciona robusta (0.01094, 0.01811) and Styela clava (0.01919, 0.03097), respectively.

A Tajima neutrality test produced negative values for all three species, but these values were significant only in the Ciona savignyi population, indicating that there was an excess of low-frequency polymorphisms, and the Ciona savignyi population was expanding. However, in the Ciona robusta/Styela clava populations, the values were not statistically significant, indicating that these two species populations did not deviate from the neutral expectations. Similarly, for Fu and Li's D* statistic, negative values were observed in all three species. The values from Ciona robusta and Ciona savignyi were statistically significant, whereas those from Styela clava were not. The results from these two analytical approaches indicate that the population of Ciona savignyi is undergoing positive selection and expansion.

Bhattachan et al. divided the three Ascidian species populations into native and invasive groups, with populations located within eastern Asian countries-like China, Japan, and Korea being considered as native groups. Since these species are believed to have originated from this region while the rest of the populations from other regions were grouped as invasive populations. Network analysis revealed that there were three haplogroups (1, 2, and 3) in Ciona robusta and Ciona savignyi, respectively. No haplogroups were found for Styela clava. In the Ciona robusta network, Bhattachan et al. found native populations in haplogroup 1, and haplogroup 3 consisted of invasive populations. On the other hand, haplogroup 2 was comprised mainly of native populations, including those from China, but few haplotypes were shared from invasive populations as well. Haplogroups 1 and 2 were connected with haplogroup 3. Similarly, in the Ciona savignyi network, Bhattachan et al. found native populations in haplogroup 1, but haplogroup 2 was entirely composed of only native populations, and haplogroup 3 consisted only of invasive populations. By contrast, there were no haplogroups present in the Styela clava network, and all haplotypes from both native and invasive populations, including those from China, were connected to each other.

Bhattachan et al. also performed a hierarchical analysis of molecular variance using cox1 haplotypes from both native and invasive populations of the three Ascidian species. There was no clear structure between native and invasive populations in Ciona robusta and Ciona savignyi, but these values were not statistically significant. In addition, we recorded a negative value for Styela clava, indicating that there was no population differentiation. By contrast, among populations of Ciona robusta, Ciona savignyi, and Styela clava, there were significant variations, with the highest level of variation appearing in Ciona savignyi. Surprisingly, within these variations, the highest value was recorded for Styela clava (77.37%),  followed by Ciona savignyi (22.77%) and Ciona robusta (21.07%).

Bhattachan et al. identified three Ascidian species from Northeast China using both morphological characteristics and genetic marker analysis. The tunic of Ciona spp. is soft and semi-transparent, whereas that of Styela clava is relatively rough and opaque. Since the tunic is mainly composed of a cellulose-like material resembling that of plants, we assume that tunic composition varies among different species. In addition, Ciona spp. absorb more water, as demonstrated by dry tunic weight, and potentially as a result, this organ became semi-transparent in nature. Furthermore, Ciona robusta is comparatively larger in size than Ciona savignyi. Recently, it was also revealed that the morpho-physiological properties play an essential role in the control of size between these two Ascidians. Hence, Bhattachan et al. use these characters to distinguish between them. It is also interesting to note that there is a red coloration at the tip of the sperm duct in Ciona robusta, which is absent in Ciona savignyi. The evolutionary and functional property of this pigmentation is not yet known. Strikingly, egg morphology also varies among these three species. For instance, long follicle cells are present on the outer covering of Ciona robusta eggs, comparatively shorter follicular cells overlay Ciona savignyi eggs, and no outer follicle cells are present on Styela clava eggs. Generally, the Ascidian egg consists of two layers of follicle cells, with a vitelline coat next to the egg membrane and several test cells between them. These outer follicle cells are vacuolated and elongated and are speculated to provide buoyancy to eggs in seawater. This may help Ascidian eggs disperse by the water current and thereby be transported to distant places. Follicle cells are also the first contact of sperm entry, and it is widely known that they function to prevent self-fertilization via a chemical reaction. Long follicle cells might have enabled a higher dispersal rate of Ciona robusta. This characteristic might also inhibit more self-fertilization in comparison to Ciona savignyi and Styela clava.

The genetic marker cox1 has been widely used for identification and characterization of genetic diversity. On the basis of barcode region of the cox1 gene from these three Ascidian species as well as other available sequences in the databases, Bhattachan et al. constructed the phylogenetic trees to infer their identification, which showed that the Ciona spp. from China was closely related to native populations, mostly from Korea to Japan. This result indicates that the Ciona spp. samples collected here from China are indeed native Ascidians, and these were not introduced from other geographical areas. However, Styela clava formed a clade with invasive populations. Bhattachan et al. also found that some haplotypes from invasive populations formed a cluster with native populations. This result indicates that there was incursion of native and invasive Ascidian populations to different parts of the world. A similar phylogenetic method was used for Ascidian identification in other geographical regions as well.

Ascidians are marine organisms with a relatively high level of genetic diversity, and there exist differences in levels of genetic diversity among the Ascidians themselves. How these differing levels of genetic diversity are maintained remains unknown. Bhattachan et al.'s current analyses confirmed that these Ascidians have a high level of genetic diversity, with Ciona savignyi exhibiting a comparatively high level of genetic diversity at the molecular level. One possible explanation might be that Ciona savignyi has a large effective population size, with differing life-history traits compared to Ciona robusta and Styela clava. Of note, a previous genome-wide study also revealed that Ciona savignyi exhibited the highest level of genetic diversity. Other comparative studies on Ascidians also confirmed that they have different evolutionary rates. This could be another reason causing the different levels of genetic diversity among these three species. In addition, the neutrality tests showed that Ciona robusta and Styela clava are undergoing neutral evolution, and Ciona savignyi is experiencing population expansion and positive selection. This also explains why Ciona savignyi exhibits a higher level of genetic diversity compared with Ciona robusta and Styela clava. Given the widespread distribution of Ascidians, it is possible to exhibit high genetic diversity across populations. This kind of observation is also seen in a wide range of other organisms.

Another important characteristic feature of Ascidians is their invasive potential. Some Ascidian species are dispersed to different geographical or ecological niches because of both anthropogenic and natural causes and are considered as invasive species. Bhattachan et al. compared the global cox1 haplotypes of these three Ascidians to understand their connectivity and population genetic structure. Global haplotypes were divided into native and invasive populations. The network analysis indicated that Ciona spp. formed haplogroups with separate native and invasive populations, although some haplotypes were shared. However, in the network of Styela clava, there was no such haplogroup formation as all of its haplotypes were interconnected, suggesting extensive incursion for this species in different geographical areas. A previous global study of Styela clava also suggested its extensive incursion, in which it was categorized as invasive species. In addition, a regional study of this species indicated the multiple sources of incursions. The results of the hierarchical analysis of molecular variance of the three species of Ascidian were also consistent with the network analysis. Bhattachan et al. found a weak population genetic structure in Ciona spp. and less genetic differentiation in Styela clava populations. An occasional gene flow between native and invasive populations of Ascidians might have occurred previously, most likely via ship transport. Bhattachan et al. clearly show that the Ciona robusta and Styela clava invasive potential is attributed to the neutral genetic diversity, whereas the invasive potential of Ciona savignyi might not be due to neutral evolution, but rather by population expansion and positive selection. Previous work indicated that a neutral force plays a role in the biological invasion and subsequent structuring of a population, but equally natural selection within biological invasion was also well characterised. It is worth noting that our analysis was based on the small sample size, because of the fewer collection sites. Increase of collection sites and sample sizes could be more accurate for the population genetic evaluation, but would not change the conclusion. Bhattachan et al.'s study reveals a global relationship between native and invasive populations and has implications in understanding the invasive potential of these three species. Thus, their work provides approaches useful for risk evaluation and management of invasive species.

See also...

Follow Sciency Thoughts on Facebook.