Ascidians collected during the Spanish Antarctic expedition CIEMAR 99/00 in the Bransfield and Gerlache Straits

During the Spanish expedition CIEMAR 99/00, on board the BIO‐Hespérides, 30 species of ascidians (distributed in 13 genera and eight families) were collected using a rock dredge in the Bransfield and Gerlache straits. Most of them were already known, but one species, Tetrazona ciemari sp. nov., is new for science. Additional morphological data are provided for six species. Five are reported for only the second time and the known distributions of five species are extended.


Introduction
The Antarctic marine fauna is relatively well known due to several expeditions, among which we may emphasize those of the Challenger (1872-1876), Pourquoi Pas? (1908)(1909)(1910), Discovery (first third of the 20th century) and Polarsten (end of the 20th century). We can also cite the Spanish expeditions on board Las Palmas (several trips between 1988 and 1991) and the Hespérides (some expeditions from 1994 to the present). There is an abundant bibliography about the Antarctic ascidian fauna, a consequence of those oceanographic cruises, but the most important papers are those by Kott (1969Kott ( , 1971 and . Among the most recent publications we may highlight those by Tatián et al. (2005), Ramos-Esplá et al. (2005) and Arntz et al. (2006). However, the Antarctic Region has been unequally studied, the best sampled areas being the Antarctic Peninsula and the South Shetland Islands, with 75 species described so far (Primo 2006), and especially Potter Cove (Sahade et al. 1998;Kowalke 1999;Kowalke et al 2001;Tatián et al. 1988aTatián et al. , 1998bTatián et al. , 2002. During the Spanish CIEMAR 99/00 expedition on-board RV Hespérides, the macrobenthos was sampled from the Bransfield and Gerlache Straits. The focus of this work was to study the ascidian fauna collected on this expedition.

Materials and methods
During the CIEMAR 99/00 expedition (December 1999), ascidians were collected at 14 stations in the Bransfield Strait using a rock dredge with 80 cm and 30 cm horizontal and vertical openings, and 10 mm mesh size ( Figure 1; Table I). We did not find ascidians at the stations situated in Bahia Sur, Livingston Island; these stations were the shallowest (3-19 m depth) and the absence of ascidians could be due to the action of sea ice as well as the mechanical action of macroalgae movement by currents and waves .
Despite animals being anaesthetized with menthol crystals, many of them were very contracted when removed from the dredges and arrived on board in a bad state. All material was preserved in 4% formaldehyde in sea water. Taxonomic determinations were based on examination of external and internal morphological characters and on the descriptions and reports of Sluiter (1906), Millar (1960), Kott (1969Kott ( , 1971), Monniot and  Monniot ( , 1994, and Sanamyan and Sanamyan (2002). The given height for solitary ascidians expresses the length from the oral siphon to the base; in the colonies it is the maximum length for encrusting colonies and the length from the base to the upper part of the colony for massive colonies. Many specimens were also compared to specimens deposited in the Antarctic ascidian collection of the Muséum National d'Histoire Naturelle of Paris (MNHN hereinafter).
The holotype of the new species is deposited at Museo Nacional de Ciencias Naturales de Madrid. All other material is deposited at the Departamento de Ecoloxia e Bioloxía Animal, Universidade de Vigo.

Results and discussion
A total of 30 species, distributed in 13 genera and eight families, was identified.

Distribution
New record: Bransfield Strait (Station B1: one specimen). Previously recorded: from the Antarctic Peninsula to Commonwealth Bay ; South Shetland  Islands Ramos-Esplá et al. 2005) and South Orkney Islands Ramos-Esplá et al. 2005). From 27 to 644 m depth.

Description
The colony (28 mm height) has grown enclosing the remnants of an algal holdfast and stalk, that maintains the colony raised over the bottom. It consists of two hemispherical heads on a globular mass (Figure 4). The tunic is translucent and of a pale pink colour; surface smooth but firm. Colourless zooids only present in the two hemispherical heads (although the postabdomens occupy the whole colony); not arranged in systems.
Zooids up to 12 mm, although they are much contracted. Oral siphon with the usual six branchial lobes; small atrial aperture surmounted by a small tri-lobed languet. We found 14 rows of stigmata (over 15 stigmata per semi-row); posterior dorsal languets longer than the anterior ones. The gut starts with a short oesophagus; the stomach is flattened but large, with more than 25 fine folds, sometimes interrupted. The gut loop forms a closed curve. Most of the zooids are males, with testes arranged in a single row only in the cardiac extreme of the postabdomen. In the female zooids, the ovary is located about the middle of the postabdomen. We also found both types of gonads simultaneously ( Figure 5).

Remarks
The holotype is lost and there is a great variability of characters in the different descriptions, especially in the colony form and the arrangement of the zooids. The type description by Harant and Vernières (1938) is imprecise and the figures are not clear. They mention strangulation between thorax and abdomen and the presence of mineral particles in the branchial sac, characteristics not mentioned again nor observed here. They did not describe the gonads either. Kott (1969) described a stalked colony, dark zooids and the simultaneous presence of testis and ovary without drawing them.  described double-row systems of zooids and accessory denticles in the oral siphon, together with the alternation of male and female gonads. However, this species is well defined by the presence of more than 20 stomach folds and the particular position of the testis. Also, we want to point out that we found both the alternative presence of male and female gonads in some specimens, and the simultaneous occurrence of both types of gonads in others.

Description
Colonies from 11 to 46 mm height. The description agrees with that of Monniot and Gaill (1978).

Remarks
Aplidium meridianum was synonymized with several species, although none of these synonymies has been retained (Monniot F 1978). The most similar species is Aplidium falklandikum Millar 1960, but the shape of the colony and the arrangement of zooids are different.

Description
Globular colonies from 17 to 25 mm height ( Figure 6a) with incrusted sand in the external surface of the test; circular systems hardly visible. Small zooids (6-7 mm) with a six-lobed branchial aperture and a tubular atrial aperture with a simple or slightly lobed languet. The branchial sac is formed by 12-13 rows of stigmata. Asymmetrical stomach with five or six poorly marked longitudinal folds. Short postabdomen (3-4 mm), sometimes with short and numerous vascular processes at the cardiac end. Most zooids are immature, but some of them have cluster-grouped testes in the upper part of the postabdomen (Figure 6b).

Remarks
This is the second record of this species since its description by Monniot and Monniot (1994). After re-examining the holotype deposited at the MNHN (registration number A1 APL.B 295), we can conclude that our specimens are juvenile colonies of this species. Although colonies (17-25 mm versus 45 mm height) and zooids (6-7 mm versus 11 mm length) are shorter, they agree in several significant characters: tunic, systems, cloacal languet, number of rows of stigmata and gonads. Some differences from the holotype (pink soft cushion-shaped colonies, six to seven incomplete stomach folds) may be attributed to the juvenile condition of our specimens. The most important difference is the lack in our specimens of parastigmatic vessels, which may be due to the great contraction of the branchial sac and/or the state of development of the zooids.

Description
Globular colonies (55 and 85 mm height) with a wide short stalk totally covered with sand. Dark orange tunic. Zooids about 15 mm length with a six-lobed branchial siphon and a simple or tri-lobed atrial languet. We found 17 rows of stigmata with eight or ten stigmata per hemirow; there is a region of non-perforated membrane at both sides of the endostyle (Figure 7b). At the base of the thorax is a structure, possibly formed by reserve material, as observed in other Aplidium species. Stomach with five poorly marked folds. Ovaries below the gut loop; testes, both grouped or arranged in rows, only in the first half of the postabdomen. Characteristic larvae incubated in the tunic with three adhesive papillae, two of them closer in the dorsal region and the third more separate in the ventral region.

Remarks
This is the first record of this species since its description by . Although there is no doubt about the identification because of its characteristic larva and the shape and coloration of the colony, we have found some characteristics not mentioned in the holotype description. Firstly, the zooid size (15 mm) is smaller and the number of rows of stigmata (17) is fewer here, despite the colony sizes being similar, but this may be within the range of variation of the species. On the other hand, we observed a portion of non-perforated membrane at both sides of the endostyle (Figure 7), a character not found by .
Flattened colonies (from 15 to 97 mm), with dense sand over the surface and throughout the test. Zooids are not visible, but their location is marked by an elevation of the tunic; they form double-row systems. Zooids up to 5 mm, closely surrounded by the tunic. The oral siphon has eight pointed lobes while the atrial one has a narrow but thick tri-lobed languet. The branchial sac has 10-12 rows of stigmata, with quite high transverse vessels. The gut loop forms a much closed curve and the stomach has five well-marked folds. Unisexual zooids with the gonads immediately below the gut loop; they occupy most of the short postabdomen (Figure 8a, b).
We have considered as uncertain some samples that show variations with regard to the above description: oral siphon with only six lobes, low transverse vessels, longer oesophagus, and more marked stomach folds ( Figure 8c).

Remarks
Aplidium radiatum closely resembles Aplidium circumvolutum (Sluiter, 1900) and Aplidium imbutum . Aplidium imbutum is clearly distinguished by a thoracic filiform extension. However, the descriptions of A. circumvolutum and A. radiatum are practically identical. The absence of visible systems in the first one may be a consequence of the high density of incrusted sand, the difference in the number of oral lobes is not sufficient to separate the two species and, although most descriptions of A. circumvolutum mention the simultaneous presence of male and female gonads, Kott (1969) suggests that this may occur when gonads are not fully developed. The main problem is that very different descriptions have been provided for A. circumvolutum; the type is lost and the few fragments of colonies (re-examined in the MNHN of Paris (registration number A1 SID-B 12 and A1 SID-B 13)) do not show the characteristics of the species. Even more, the holotype description of A. radiatum does not agree with the specimens to which it is referred (Monniot F 1978).  ( Figure 9) Ritterella mirifica .

Distribution
New record: Bransfield Strait (Station B7: one specimen). Previously recorded: Ross Sea ; South Shetland Islands . From 142 to 365 m depth.

Description
We found only one very damaged stalked colony (50 mm height). Zooids with tubular siphons; the atrial one has numerous lobes ( Figure 9b). Twelve to 13 rows of stigmata in the branchial sac. Stomach with five or six longitudinal folds. Testes slightly below the gut loop ( Figure 9a). It was impossible to extract a complete postabdomen, so the length of zooids is unknown.

Remarks
The only Antarctic species of the genus Ritterella is R. mirifica, the paratype of which has been re-examined at the MNHN (MNHN A1 RIT 5). The characteristic atrial siphon, the grouped testes slightly below the gut loop, and the number of rows of stigmata are similar. The shape of the colonies is rather different (stalked here, globular in the holotype), but in both cases they are quite damaged.

Description
Although most of the specimens are immature, the colonies (from 18 to 230 mm height) have the characteristic aspect of the species: stalk with incrusted sand and rounded head, circular systems of zooids.

Remarks
Morphology of our specimens coincides with those described by F. .

Description
Small colonies (up to 31 mm in our specimens) with basal processes (Figure 10a) to attach to substrata. Few systems of zooids (usually one) per colony; small zooids (about 5 mm) with few rows of stigmata (15 in our specimens) and gonads a little separated from the gut loop ( Figure 10b).

Remarks
Frequently confused with Synoicum adareanum (Herdman, 1902). After re-examining the holotype (no. 489 T) we conclude that, despite the great variety of shapes, our specimens belong to this species because of the few zooid systems, the root-like processes of the colony, the small size of zooids, few rows of stigmata and the location of the gonads.

Distribution
New record: Bransfield Strait (Station B6: four specimens). Previously recorded: Wilkes Land ; South Shetland Islands ; Balleny and South Orkney Islands . From 0 to 350 m depth.

Description
Stalked colonies (from 19 to 112 mm height) with a slight constriction between head and stalk ( Figure 11a). Circular systems. About 20 rows of branchial stigmata with 20 stigmata per half-row ( Figure 11b). Zooids are immature.

Remarks
Our specimens coincide with the holotype described by  except for few stigmata per half row (20 in our specimens versus 35 in the holotype). Synoicum ostentor is close to Synoicum adareanum, but S. ostentor has a constriction between head and stalk and a more marked difference in the texture of both parts. Zooids are similar size, but differ in a greater number of rows of stigmata, and stigmata per row, and greater distance between abdomen and ovary in S. ostentor. This is the second record of this species since its description by .

Description
Cylindrical colonies up to 30 mm with irregular basal processes (Figure 12a). Strong tunic with embedded sand. One system per colony with the common cloacal opening in the upper part. Zooids up to 15 mm. Long branchial siphon with six lobes; tubular atrial siphon with three lobes from the dorsal border and three minute lobes more from the ventral border (Figure 12b). Branchial sac with 14 rows of stigmata. Initial constriction of the oesophagus (hardly visible because of the great contraction of the animals). Smooth, large, rounded stomach. The gut loop does not show torsion. All the specimens are immature.

Remarks
The description agrees with the holotype re-examined at MNHN (registration number A1 711-716 and A1 SYN-29) (the atrial siphon, and the constriction of the oesophagus being characteristic) except for the fact that in this case the whole colony is incrusted with sand and is not naked on the upper part. The zooids are shorter, but this may be due to the absence of gonads.
Synoicum polygyna is similar to Synoicum ramulosum Kott, 1969. The only differences are smaller colonies and zooids, fewer rows of stigmata and absence of constriction of the oesophagus in S. ramulosum. It could be possible that Kott's specimens were juveniles of S. polygyna.

Description
The colony (28 mm height) has a curious cylindrical form, attached to small rocks by its basal half, with two extensions (probably common cloacal openings) at the top, so that it could be confused with a solitary ascidian (Figure 13). Tunic is soft, pink, with some incrusted sand.
The small zooids (up to 7 mm) have a six-lobed oral siphon and a tubular cloacal one with a dorsal tri-lobed languet (Figure 14). The thorax is much contracted and it contains a lot of reserve material, so it was impossible to count the number of rows of stigmata. The stomach is smooth-walled and the gut seems not to be differentiated. All of them are immature.

Remarks
Because of the low number of zooids in the colony and their bad preservation it was impossible to identify them to species level.

Description
Small colonies (up to 24 mm of maximum length), rounded but flattened ( Figure 15). The tunic is rather soft, resistant and practically free from sand, except for some dispersed particles inserted irregularly among the zooids. The zooids are scattered without apparent order for the whole colony.
The zooids measure 4 mm on average, the abdomen being a little shorter than the thorax. Both siphons are tubular and six-lobed, the atrial one slightly longer. They are located at the same level on the dorsal part. There are about 20 simple branchial tentacles of several sizes. The branchial sac has four rows of about 20 stigmata per semi-row, without parastigmatic vessels (Figure 16b). The first two rows are smaller. The languets of the dorsal lamina are long and pointed. There are no developed gonads, but we observed the sperm duct in the lower part of the thorax of some zooids and the presence of small oocytes in the basal part of the abdomen (Figure 16a). There are no larvae.

Remarks
The genus Tetrazona was created by Michaelsen (1930) to include those species close to Cystodytes, but without calcareous spicules. Currently, only two species of this genus are known: T. porrecta Millar, 1962 andT. glareosa (Sluiter, 1906).
Tetrazona porrecta is a South African species that differs both in the colony structure (upright, long and narrow, with irregular lobes and protuberances) and in the zooids, with a similar size but with an abdomen longer than the thorax, fewer stigmata, folds on the stomach wall and gonads immediately below the lower bend of the gut loop. Furthermore, we have had access to a specimen from South Africa (collected by the Coral Reef Research Foundation), so we can affirm that this is not the same species.
On the other hand, we examined some specimens deposited at the MNHN of the Antarctic species T. glareosa, and it presents a similar structure of the colony. However, there are important differences. First, T. glareosa has asterisk-like spicules irregularly scattered in the tunic; although Kott (1969) commented that these spicules, described by Sluiter (1906) as siliceous, could be artefacts of preservation,  and Tatián et al. (2005) found this species again and described calcareous spicules. Another significant difference is the size of the zooids, up to 2 mm, which seems to be a constant character independently of the colony size in T. glareosa, and up to 4 mm in our specimens. Also, while in T. glareosa the proportion thorax/abdomen is similar, in our specimens the thorax is always longer, sometimes twice the size of the abdomen. Another difference is the number of oral tentacles: 12 in the specimens of T. glareosa described by Sluiter (1906) and only four in those described by , short and pointed in both cases, while we have found 20 longer oral tentacles. As for the gut loop, there is no net transition between stomach and intestine in T. glareosa, whereas it exists in our specimens. Finally,  mentioned long postabdominal vascular appendix, crossed in every direction in the tunic; however, instead of these prolongations, we found a relatively long vascular appendix close to the gut loop.

Description
Our specimen agrees with the description by Kott (1969).

Remarks
Three Tylobranchion Antarctic species were described in function of their different size, stomach folds, and the shape of musculature and papillae (Arnback-Christie-Linde 1950). They were synonymized by Kott (1954), supported by Millar (1960) who found that these characters overlap.  considered the problem still unresolved, but they admitted that these differences might be due to age or physiological stage of the colony.

Description
Our specimens coincide with those described by Kott (1969).

Remarks
Although species of the genus Ascidia can be difficult to distinguish, only three species are recognized from the Antarctic Region. Ascidia translucida Herdman, 1880 is clearly distinct because of its size (see Table II), and because the dorsal tubercle fills the peritubercular area and is very convoluted, while Ascidia challengeri Herdman, 1882 and Ascidia meridionalis Herdman, 1880 are more similar. We summarize distinguishing characters in Table II.

Description
This species, with transparent, soft and smooth tunic and four branchial folds, is easily recognizable by the gonad structure. There are two on each side of the body, formed by a tubular ovary with numerous short biramous branches; the testis follicles are intimately associated with the tips of these branches. The sperm ducts join the vas deferens on the medial surface of the ovary, running parallel to the oviduct.

Remarks
Although it has sometimes been confused with Styela paessleri Michaelsen, 1898, the gonad structure is quite different since testis and ovary are not enclosed in a common envelope.

Remarks
Cnemidocarpa verrucosa is easy to identify through the tunic that, although it can be very variable, is resistant and opaque (generally light pink or yellowish), but thin and flexible. The surface is covered by papillae (with spines in the young specimens). Other characters such as the body wall close to the tunic but separated, and four poorly marked branchial folds are characteristic of the species too. Two gonads on each side; one on the left is between body wall and gut loop. This species clearly differs from C. pfefferi because of the biramous branches of the ovaries of the last one.

Remarks
Our specimens agree with the description by Kott (1969) under the name of Styela insinuosa. The genus Dicarpa, created by Millar (1955b), was initially described for only one species, D. simplex, the distinctive characters being: four-lobed siphons, endocarps in the whole body wall, simple oral tentacles, smooth dorsal lamina, three to four branchial folds and one Polycarpa-like gonad on each side. Later, more species were included and the characters that define the genus are the type of branchia and the presence of only one gonad on each side, although the morphology is variable. Dicarpa insinuosa has a tubular ovary and several groups of testicular follicles surrounding the ovary, especially abundant at the posterior end.

Description
Ovoid shape, 17 mm high and 9 mm across at the widest point. The lower part of the body narrows towards the base, which is covered by test hairs with a little adhered sand. No test hairs or foreign particles are present on the rest of the body, which is yellowish pink, smooth, very thin but tough.
Siphons, short, four-lobed, both dorsal and close to each other. There are over 32 simple branchial tentacles, of two sizes. The small dorsal tubercle is a simple oval transverse slit. The dorsal lamina, with a plain margin, is wider on the posterior part. The branchial sac has three longitudinal vessels on each side; up to 30 stigmata per mesh, with parastigmatic vessels. A short oesophagus is followed by the stomach, with internal folds and a small pyloric caecum. The intestine forms a closed gut loop after the stomach, and then bends anteriorly near the oesophagus. The anus has eight well-marked lobes. Only one gonad has been found, mainly situated on the left side, but it crosses the endostyle line, thus also occupying the right side ( Figure 17). The testes are located on the posterior side of the ovary. They are long narrow follicles arranged in a series along the length of the gonad. There are two long sperm ducts, one on each side of the body, projecting from both ends of the gonad, but not terminal. The oviducal opening was not observed.

Remarks
Our specimen is the largest collected up to now and agrees perfectly with the description of the holotype except in small details such as external appearance of the tunic-covered with wart-like swellings according to Millar (1960)-or the number of oral tentacles, which probably depends on the size. Monniot and Monniot (1980) described one gonad on each side, with the same general structure. The presence of two sperm ducts in the specimens with only one gonad may be a sign of the fusion of two initial gonads.

Remarks
In general our specimens agree with the description by Kott (1969). They have two gonads on each side although one specimen has a single gonad on the left side. Characteristic pointed already by Kott (1969).

Description
One ovoid colony, 21 mm height. The test is thin, but firm and tough. The surface is covered with minute papillae which contain, in the dorsal half of the body, calcareous spicules, typical of the species. Each spicule has numerous short spines in a slanting plane; near the siphons, the spicules are arranged with the oblique plane toward the siphon apertures. In the middle line of the body may be found a regular series of spine sizes, increasingly larger ones higher up.
Atrial and branchial siphons are almost sessile, bilabiate and directed anteriorly. There are 33 branched branchial tentacles of three sizes. The branchial sac has six very high folds on each side. The right gonad has split into two, although the ovaries are still fused together. The larger gonad has a double gonoduct ( Figure 18). There is a large testis lobe on the distal end of each ovary and smaller lobes along it, arranged without order.

Description
Up to 38 mm plus a stalk of 243 mm.
The characteristics that separate P. bouvetensis from the other species are: globular body, long stalk located over a third of the ventral face at the level of the gut loop, tunic with variable small spines, weak musculature, right gonad divided in two to nine lobes, left gonad in the gut loop, endocarps over the gonads, quite small parietal organs distant from gonads and gut, and no atrial organs. Some of these characteristics are not exclusive to the species, but as a whole they are definitive.

Remarks
This is a common Antarctic species with a characteristic aspect. The most significant character is the presence of a strong membrane at the base of both siphons, possibly a sphincter to close the openings, maybe as a defence mechanism (Kott 1969). Other important characteristics are: poorly developed seventh branchial fold, arborescent liver, and Polycarpa-like gonads.

Description
We found one colony 98 mm height plus a stalk of 45 mm. The tunic is thick and fleshy. The branchial siphon is directed downward and the atrial one, sessile, is just above. The dorsal tubercle is a double cone and the horns, both of them forming a spiral towards the right, have a convoluted course. One gonad on each side. There are two endocarps that cover the right gonad and the gut together with the left gonad, respectively. The gonoducts are very long and they open near the atrial siphon.

Remarks
Although variable in external appearance, this large species has a series of distinctive characteristics. We agree with  that there is only one gonad on each side and that probably Kott (1969) confused the heart, well developed and covered with an endocarp too, with a second right gonad.

Remarks
The description of our specimens agrees with that of , except for the specimens from Station B3, both small (up to 20 mm) and very damaged. Although the external characteristics coincide, one of the specimens has eight branchial folds on the left side (on the right it has the six branchial folds characteristic of the species), the fourth being not well developed. The other one is internally smashed and no structure can be recognized.

Remarks
Identification of several Antarctic Molgula species, especially small-sized species and frequently covered with sand, is difficult without a meticulous examination.
The two species found in the present collection, Molgula hodgsoni and M. pedunculata, can be differentiated by several characteristics summarized in Table III. Some untailed embryos were observed incubating in the atrial cavity.

Remarks
Untailed embryos were present in the atrial cavity.