First record of Styela clava ( Tunicata , Ascidiacea ) in the Mediterranean region

The solitary ascidian Styela clava Herdman, 1882 is recorded for the first time in the Mediterranean region. It was found in the Bassin de Thau, near Sète; it has not yet been found in the Mediterranean Sea. The Bassin de Thau specimens are described and compared with Northern European specimens. The proximity of commercial shellfisheries to the discovered populations, and the absence of S. clava from other harbours and marinas along the coast, suggests that the species may have been introduced by shellfish transfer.


Introduction
The solitary ascidian Styela clava Herdman, 1882 is native to the north-west Pacific, particularly Japanese and Korean waters.It was first discovered in European waters in 1953 in the estuary of the Lynher River, near Plymouth (UK) and recorded as Styela mammiculata sp.nov.(Carlisle 1954); Millar (1960) demonstrated that this "new species" was synonymous with Styela clava Herdman, 1882.It is probable that it was introduced into Plymouth Sound by military craft returning from the northwest Pacific during the Korean War.Since its initial discovery, it has spread up the North Sea coast of Europe as far north as Denmark (Lützen 1999), and south along the Atlantic coast to Portugal (Davis and Davis 2004;2005).A full account of the current distribution is presented in Davis et al. (2007).
S. clava possesses a firm, club-shaped body that can grow up to 220 mm in total length.It is attached to the substratum by a short stolon, the base of which forms an expanded membranous plate that adheres the organism to the substratum.A solitary ascidian resembling S. clava was first observed in the Bassin de Thau, near Sète in 2004 (D.Luquet, personal communication); a sample for identification was collected by D. Luquet on June 1st, 2005.Despite extensive searches of harbours and marinas along the adjacent Mediterranean coastline, no populations could be located in the Mediterranean Sea itself.Consequently, publication of the new record was delayed until it could be established that a sustainable population of the ascidians exists in the Bassin de Thau.

Sampling site
The Bassin (or Etang) de Thau is a 21 km long and 5 km wide lagoon in southern France (Figure 1).The mean depth of the lagoon is 4.5m, but the depth can exceed 30m in places.The tidal range is small, generally less than 30 cm in amplitude (La Jeunesse and Elliot 2004), and the tidal currents are weak (Souchu et al. 2001).The 7,500 hectares expanse of brackish water is separated from the Mediterranean Sea by a narrow sand bar with openings at Marseillan-Plage, the Pisse-Saumes canal, and at Sète, the Quilles canal (Figure 2).The Bassin de Thau is also connected to the Mediterranean Sea by the Canal de Sète, which flows through the eastern edge of the town of Sète.
Six specimens of the solitary ascidian were collected, by scuba diving, from rocks at ~5m depth in the southern end of the Bassin de Thau (approximately 43°25.6'N03°39.5'W)by David Luquet (L'Observatoire Océanologique de Villefranche-sur-Mer) on June 1st, 2005 and preserved in 10% formaldehyde-seawater solution for subsequent examination (Figure 3).

Description of the specimens
The specimens exhibited the same general morphology as Styela clava that we have found in Northern European waters.The individuals collected ranged from 65 mm to 145 mm total length.Each had a firm club-shaped body, a short stem-like stolon and a membranous basal plate.The orange-brown tunic on the body and stolon had longitudinal grooves; the anterior tunic also had lateral grooves, which produced a mammillated pattern.The siphons were anterior facing, close together, raised and tapered; they were distally marked with four dark brown stripes alternating with narrower pale stripes.The oral siphon was terminal and the atrial siphon was set close by on the dorsal surface.
Internal examination of three of the specimens (100 mm, 125 mm and 130 mm total length) revealed the stomach was in the descending portion of the intestinal tract and the intestine curved upwards towards the atrial siphon.There were more sinuous ovaries on the right side (5, 7 and 7) than on the left (3, 3 and 3), and the numerous testicular follicles were clustered together along the ovaries to form white lobes.The gut and gonad descriptions are characteristic   1) indicated that these specimens were indeed S. clava.(Minchin and Duggan 1988), or attached to ships' hulls (Gollasch 2002) including the hulls of pleasure craft, or in sea chests (Davis and Davis 2004;Coutts and Dodgshun 2007).
Hydrodynamic analysis indicates that hull transport is unlikely because any S. clava larvae that do settle and develop on the external hull would be unlikely to survive the sustained highvelocity movement through water when the ship is in service.Fouling organisms may be transported on ships' hulls if they are attached in areas of reduced flow such as sea-chests, bowthruster tubes, stabiliser pockets and the cavities around the stern tubes and propellers, but large commercial ships do not enter the Bassin de Thau.Therefore, if S. clava arrived in the area as hull fouling, the most likely points of entry would be the military port of Toulon (which has shipping links with Brest and Cherbourg where S. clava populations flourish) and the commercial port of Marseilles.No specimens of S. clava could be found in marinas adjacent to these ports; marinas were surveyed because they provide easy access to permanently submerged hard substrate in a sheltered environment.Despite exhaustive searches, using the methods described above, no specimens of S. clava have been found in any northern Mediterranean ports or marinas (Figure 1).
Long distance dispersal of S. clava could occur if juvenile animals were transported attached to oyster shells when the oysters were re-laid.It is probable that this was the vector by which S. clava was introduced into the Limfjord (Lützen 1999) and Prince Edward Island (Locke et al. 2007).The Bassin de Thau has a thriving commercial shellfishery, the most important product being oysters -mainly Crassostrea gigas (Thunberg, 1793).There are approximately 750 oyster farmers in the lagoon, producing approximately 13,000 tonnes of oysters annually.C. gigas was imported from Japan to the Bassin de Thau from the late 1960s until at least the mid 1970s; however, it is possible that some juvenile oysters have been imported during the last few years and grown to marketable size.

Significance of the new record
In Prince Edward Island, Canada, Styela clava is a major pest to the mussel farming industry (Thompson and MacNair 2004).It settles on the mussel ropes and competes with the mussels for food, reducing mussel production by up to 40% and costing the mussel industry several million C$ per year.The recent discovery of S. clava in New Zealand (Davis and Davis 2006) caused considerable alarm; the green lipped mussel industry in New Zealand is worth an estimated NZ$ 150 million per year and is now considered to be at risk.
Many species of shellfish are farmed commercially in the Bassin de Thau, the most important being oysters.There are many similarities between oyster farming in the Bassin de Thau and mussel farming in Prince Edward Island, so the threat from S. clava should be similar.However, the two sites differ in the availability of suitable settlement substrate, a major resource limitation for growth of S. clava populations.The natural substrate available in Prince Edward Island is mainly soft, typically mud (Bourque et al. 2007), so S. clava exploits the mussel farming equipment.The abundant hard substrate in the Bassin de Thau may mitigate the threat of S. clava to oyster production.

Potential for further spread of Styela clava
The most important prerequisite for Styela clava to spread from one site to another is that the donor and receiving ecosystems are connected by a dispersal vector.Within the lagoon, S. clava will spread by natural larvae dispersion; the animals are free spawners so planktonic eggs and larvae can be carried to new sites by horizontal currents.In addition, drifting flotsam can carry attached juveniles and adults to new sites.Spread outside the lagoon is likely to be the result of larvae dispersion through the canals at Marseillan-Plage and Sète, possibly with sublittoral "stepping stone" populations established on suitable bottom substrate, although flotsam and man-aided transport of adult animals into the Mediterranean are possible.In either case, successful establishment depends on the receiving habitat being suitable; the key criteria appear to be salinity between 22 and 34.5 psu and summer water temperature in excess of 15ºC for spawning (Bourque et al. 2007).
Spot salinities varied from 28 to 30 psu in the Bassin de Thau, and from 27 to 34.5 psu in some adjacent harbours (Annex 1).Therefore, it is possible that S. clava could become established in the Mediterranean in the Languedoc-Roussillon region.However, extensive range expansion is unlikely as salinity exceeded 34.5 psu in many of the Mediterranean harbours and marinas surveyed (Annex 2).In fact, 63 Mediterranean sites were inspected (Figure 2) but no specimens of S. clava were found at any.Most of the sites with salinity greater than 22 psu supported populations of other solitary ascidians, mainly Styela plicata (Lesueur, 1823) with occasional Ciona intestinalis (L.), Molgula manhattensis (De Kay, 1843) and Ascidiella aspersa (Müller, 1776).However, it should be remembered that the inability to find specimens at a site does not necessarily mean that there are no populations present in the area.
Water temperature may be an important factor controlling population growth.In 2006, a maximum surface water temperature of 29.1ºC was recorded between Bouzigues and Sète in July (Ifremer 2007), although it only reached 24.3ºC in 2007.The maximum recorded bottom water temperatures at the site were 26.6ºC in July 2006 and 22.9ºC in August 2007 (Ifremer 2007), temperatures that occur at other sites where S. clava has been found, so S. clava may be able to survive at depth.However the effect of temperature may be exacerbated by the development of anoxic conditions in the bottom waters of the lagoon during the summer months.Mazouni et al. (1996) noted anoxia in the lagoon in July which they thought could be responsible for a massive mortality of the benthos.Indeed, Mazouni et al. (2001) reported that only young ascidians (Ciona intestinalis, Phallusia mammillata and Botryllus sp.) were present five weeks after a bottom anoxia event in 1992, although the maximum water temperature recorded was only 26ºC.Incidentally, although Mazouni et al. (2001) monitored the composition of biofouling communities in the Bassin de Thau during 1992, they did not record the presence of S. clava.

Conclusions
The solitary ascidian collected in the Bassin de Thau, near Sète, on June 1st, 2005 was identified as Styela clava.This is the first record of its presence in the Mediterranean region.The lagoon population appears to be sustainable.However, extensive searches of harbours and marinas along the adjacent Mediterranean coastline did not reveal any populations of S. clava in the Mediterranean Sea itself.
The proximity of commercial shellfisheries to the discovered populations, the lack of commercial shipping routes into the lagoon and the absence of S. clava from other harbours and marinas along the coast, suggest that the species may have been introduced by shellfish transfer.

Figure 1 .
Figure 1.Location of the Bassin de Thau and Mediterranean sampling sites.Asterisk -Styela clava present (Bassin de Thau), open cycles -Styela clava absent (adjacent sites are included in a single symbol), filled cycles -nearest recorded Styela clava populations

Figure 2 .
Figure 2. The Bassin de Thau showing the sites where Styela clava was found

Table 1 .
Summary of the distinguishing features of Styela clava specimens