On some frenulate species ( Annelida : Polychaeta : Siboglinidae ) from mud volcanoes in the Gulf of Cadiz ( NE Atlantic )

Collections of Frenulata made by the rV Professor logachev in the Gulf of Cadiz in 2006 contain two new species, one belonging to the genus Spirobrachia, and another that was assigned to a new genus. Spirobrachia tripeira n. sp. is the first record of the genus in the north Atlantic ocean; it has a very stiff, straight, orange tube; its tentacles have pinnules and are arranged in a spiral crown without a lophophore. Bobmarleya gadensis n. gen. et n. sp. is characterised by an unusually long tentacular crown composed of about 40 free tentacles. it shares many characteristics with the genus Oligobrachia but the large number and extreme length of the tentacles and a combination of other characters justify the designation of a new genus. The specimens of Lamellisabella denticulata Southward, 1978 collected during the M.S. Merian 03/01 cruise provide the first record of this species in the Gulf of Cadiz.

Scientia Marina 72(2) June 2008, 361-371, Barcelona (Spain) iSSn: 0214-8358 inTroDuCTion Siboglinids (also called Pogonophora) include three groups of marine protostomes, frenulates, moniliferans and vestimentiferans. The group has a varied and complex taxonomic history in that they have been placed in both Deutorostomia (ivanov and Petrunkevitch, 1955;Southward, 1963) and Protostomia (van der land and nørrevang, 1975;South-ward, 1988) and have been assigned to all taxonomic ranks from family to phylum. The first member of this group, Siboglinum weberi was not found until early in the 20th century. Caullery (1914) named the genus Siboglinum and the family Siboglinidae, without assigning the animal to a particular phylum. The species was only described in 1944 (Caullery, 1944). Annelid affinities are now supported by both morphological and genetic studies. rouse and Fauchald (1997) con-ducted a series of cladistic analysis of the morphology of polychaetes and stated that: "The Pogonophora should now be reclassified as members of the clade Sabellida". They argued that "since the name Pogonophora was misleading at this level, the name of the group should revert to that of the first family name originally formulated for members of the group, that of Siboglinidae Caullery, 1914". This name change was also proposed by McHugh (1997), it has been used by other authors (Schulze, 2003;Halanych, 2005) and it will be used in this paper. For the taxonomy within the Siboglinidae, rouse (2001) used the names Frenulata, Monilifera and Vestimentifera for three clades. For objections to the replacement of the group name Pogonophora by Siboglinidae see Southward et al. (2005) and Bartolomaeus et al. (2005).
All siboglinids inhabit reducing environments, mostly in the deep-sea, although the levels of sulphide and methane and the type of substrate vary. Frenulates are generally found in anoxic sediments with the anterior end of the tube extending into the oxygenated bottom water. They have been found in hydrocarbon seeps, on continental slopes, and at the bottom of fjords. An increasing effort put into deepsea sampling in the Atlantic is gradually disclosing more species as well as more genera of frenulates, and reducing the apparent deficiency of genera in the Atlantic compared with the Pacific ocean.
Chemosynthetic-based communities, often dominated by frenulates, have been found in several mud volcanoes in the Gulf of Cadiz (Cunha et al., 2001;rodrigues and Cunha, 2005). Here we describe two new frenulate species collected from the Porto and Carlos ribeiro mud volcanoes during the TTr16 cruise (Training Through research Programme, ioC-unESCo) on board the rV Professor logachev, and report the first record of Lamellisabella denticulata in the Gulf of Cadiz, collected during the MSM01/03 cruise. Specimens of Siboglinum and Polybrachia were also collected from several mud volcanoes, but because further morphological and molecular studies are necessary to elucidate their taxonomy, these genera will be reported elsewhere.

Study area
For more than a decade, the international marine scientific community has deployed considerable ef-forts in studying the occurrence of mud volcanism, cold seepage and gas hydrates in the Gulf of Cadiz (Baraza and Ercilla, 1996;Pinheiro et al., 2003;Somoza et al., 2003). Since the discovery of the Gulf's first mud volcano in 1999, research cruises have steadily unveiled one mud volcano after another (Gardner, 2001;Pinheiro et al., 2003;Somoza et al., 2003;Van rensbergen et al., 2005). located in a compressional tectonic province, the mud volcanoes in the Gulf of Cadiz are more numerous than anywhere else on the European Atlantic margins. The compression between the Eurasian and African tectonic plates creates an interesting geophysical template (Sartori et al., 1994;Maldonado et al., 1999;Gutcher et al., 2002) shaped by volcanic activity and by the interaction between the topography and the circulation of the Atlantic and Mediterranean Waters (Peliz et al., 2007). Found in water depths between 200 and 4000 m, these mud volcanoes are clustered in several fields on the Portuguese, Spanish, and Moroccan margins and show considerable variations in dimension, morphology and composition of erupted material and fuids Van rensbergen et al., 2005;Hensen et al., 2007;niemann et al., 2006).

Sample collection and processing
Specimens of the two new species were obtained from TV-guided grab samples collected on the Porto mud volcano (35°33.77'n, 8°30.42'W, 3902 m depth) and the Carlos ribeiro mud volcano (35°47.23'n, 8°25.27'W, 2200 m depth), respectively. Specimens of Lamellisabella denticulata were collected on the Porto mud volcano with a uSnEl box core. The specimens were carefully picked from the sediment, and preserved in 70 or 96% ethanol (the latter will be used for future molecular studies).
All drawings were made using a camera lucida attached to a stereomicroscope. Air-dried pieces of the girdle of specimens of both species were mounted onto stubs using adhesive carbon disks, sputtercoated with gold-palladium (Polaron E500; 3x30s) and observed with a JEol JSM-5400 scanning electron microscope.
For the histological study of the internal anatomy the specimens were slowly dehydrated by transfer to 90% ethanol overnight followed by a period of 9 hours in 100% ethanol with change of solution every 3 hours. The segments were cleared with 100% Histoclear TM for 12 hours depending on the size of on FrEnulATA FroM THE GulF oF CADiz • 363 SCi. MAr., 72(2), June 2008, 361-371. iSSn 0214-8358 the segment and impregnated in paraffin wax at 70°C for 12 to 24 hours. The tissue was then embedded in wax, sectioned at 5 μm, and stained with Mayer's hematoxylin and eosin.
Diagnosis. More than 30 free tentacles packed in a regular arrangement at their base, with pinnules. Pale and thin bridle keels. Small cephalic lobe with an irregular tip. Two rows of plaqueless metameric papillae separated by a furrow. Two girdles lying on welldeveloped ridges, with the two halves of both girdles being separated dorsally by a relatively large group of papillae. The tube is soft and ringed; semi-transparent at the anterior end, brown with darker rings in the middle and white/greyish in the posterior end.
Etymology. The generic name is given as an allusion to the shape of the tentacular crown in which the tentacles largely resemble dreadlocks, a hairstyle popularised by the reggae singer and songwriter Bob Marley. Etymology. The specific name comes from the roman name of the city of Cadiz, Gades, in reference to the Gulf of Cadiz.

Bobmarleya gadensis
Diagnosis. As this genus is currently monotypic, the diagnosis is as the generic diagnosis.
Description. This is a large, rather dark frenulate, living in a soft ringed tube about 2 mm in diameter. The tube is white, semi-transparent on the first 5 to 10 mm, but it becomes dark brown in the middle and then lighter again to reach a white/greyish colour at the posterior end (Fig. 1). The rings are black, with a length equal to the diameter of the tube on its anterior portion, but on the middle section they become incomplete and closer together. on the posterior portion the rings are almost absent and the tube is smooth. The longest occupied tube was 975 mm.
The most obvious feature of this species is the long, up to 65 mm long, orange-brown tentacular crown (Fig. 4C); it consists of about 40 free tentacles arranged regularly at their base ( Fig. 5A), with two rows of pinnules along the inner side (Fig. 4A).
The forepart of the body is short (2.33 to 3.66 mm) and thick (1.40 to 1.73 mm), with a pronounced dorsal convexity ( Fig. 2A-C). The anterior end is irregular, has a mid-dorsal swelling and partially encloses the base of the tentacles ( Fig. 2A). The cephalic lobe is small and has an irregular end. Between the cephalic lobe and the bridle there is a mid-ventral patch of granular cells (Fig. 2B).
The colourless, narrow keels of the bridle lie on well-developed ridges and are not fused dorsally or ventrally. Anterior to the bridle are 2 dorso-lateral ridges (Figs. 2A-C). A mid-dorsal furrow begins behind the bridle and runs as far as the junction with the trunk ( Fig. 2A). The metameric region at the anterior end of the trunk is 8 to 11 mm long and has two wide glandular ridges, separated by a median dorsal furrow that runs along most of the length of the pre-annular region. Anteriorly the glands are not grouped, but after 1 mm they are grouped inside two rows of plaqueless papillae ( Fig. 2A). laterally there are smaller pyriform glands with rings of brown epidermal cells around their openings forming two longitudinal brown stripes (Fig. 2C); the ventral side is smooth and flat (Fig. 2B).
Posterior to the metameric region the trunk is brownish with scattered darker spots, and after the posterior end of the mid-dorsal furrow there is a region with more than 25 enlarged papillae on the dorsal side (Fig. 2E). Some of these enlarged papillae are tipped by singular cuticular plaques. The two girdles lie on well-developed ridges surrounded by rather dark pigment spots, and are separated from the enlarged papillae by a section of 5 to 11 mm. Anteriorly to the girdles starts a strip of 40 small papillae that separates dorsally the two halves of both girdles. Ventrally they are also both interrupted, but the posterior gap is very narrow (Fig. 2E and F). The chaetae of the girdles are arranged in 8 rows; they are 12 to 15 μm long and elongate in shape with two groups of teeth facing opposite directions. The anterior group is small, with smaller and very curved teeth (Fig. 5C). After the posterior girdle there is a conical enlarged papilla. The post-annular papillae are arranged in transverse rows of 5 to 7. The opisthosoma was missing in all the specimens collected.
The spermatophores are 1.6 mm long, spindleshaped with a pair of small wings at the base of the filament ( Fig. 2G and 4D).
Distribution. Known only from the type locality, Carlos ribeiro mud volcano, at a depth of 2200 m.
Remarks. The arrangement of the tentacles of this genus is unique among Frenulata. There are several other genera with more than two free tentacles (Table 1), but in all of them the base of the tentacular crown is horseshoe-shaped, although in Cyclobrachia and Zenkevitchiana the ends bend round to complete the ring (ivanov, 1963). Because histological sections to study the base of the tentacular crown were made in only one of the specimens, it is open to discussion whether this character is sufficient to create a new genus. However, a number of other characters justify the creation of a new genus. The number of tentacles corresponds to that of Polybrachia but the species of this genus are characterised by a segmented tube with funnel-like collars on each segment, whereas the species described here has a soft ringed tube similar to that of species of Oligobrachia. The structure of the spermatophore is also similar to that of Oligobrachia (Southward, 1978a). on the other hand, species of Oligobrachia are characterised by the strong bridle keels (Southward, 1978a), which are pale and narrow in the genus here described.
Bobmarleya gadensis shows a general resemblance to Birsteinia but the latter has a stiff tube and plaques on the metameric papillae (ivanov, 1952, 1963). Cuticular plaques on the metameric papillae are also present in Polybrachia, Diplobrachia, Cyclobrachia, Heptobrachia and Galathealinum.
Although size characteristics are not diagnostic of the superspecific taxa of Frenulata, it is pertinent to note the length of the tentacular crown of the genus, which is the longest ever recorded (up to 65 mm). However, in all the specimens collected the tentacles deteriorated very quickly and at the time of fixation they were no longer than 1 mm and had no pinnules in this region (Figs. 2A-C and 4B).
Genus Lamellisabella ushakov, 1933 Lamellisabella denticulata Southward, 1978 Lamellisabella denticulata Southward, 1978: 713-716, Fig. 1 and 2. Distribution. L. denticulata is the only species of Lamellisabella described from the Atlantic ocean (Southward, 1978b). it was first found in the Bay of Biscay at 4000 m depth, but Southward (1978b) proposed that its range extends along the continental rise as far south as the Gulf of Guinea. in the Gulf of Cadiz it was found on the Porto mud volcano at a depth of 3902 m. Diagnosis. Tentacular spiral consisting of 80 to 110 tentacles with a quadruple row of pinnules each. lophophore absent. Brown/orange tube, hard for most of its length.
Etymology. The specific name tripeira is given in reference to the nickname of the inhabitants of the city of Porto, which originates from the typical dish of the city, tripas.
Description. The light brown/orange tube is straight and very stiff, with thick, strong walls tapering towards the posterior end, which becomes slightly sinuous but remains thick-walled. There is  Kirkegaard, 1956 78-268 Choanophorus Bubko, 1965 18 Bobmarleya n. gen.
ca. 40 a short, transparent anterior part, which is soft and thin walled (Fig. 3A). The diameter of the tube varies from 0.57 to 2.05 mm . The tentacles (80 to 100) are arranged in a spiral crown with 5 turns (Fig. 3B-D, 4E, 5B). The spire is leotropic, its direction of turn (when viewed from the anterior end) being anti-clockwise in all the specimens examined. The tentacles adhere side by side and have pinnules in a quadruple row on the zone facing the inside of the crown. The bases of the tentacles are situated on one plane on a tablelike broadening of the forepart, without a lophophore. The whole tentacular crown is up to 22.8 mm in length.
The forepart of the body is 2 to 4 times as long as wide, the maximum length and diameter recorded being respectively 4.7 mm and 1.2 mm. The cephalic lobe is very wide and dorso-ventrally flattened ( Fig.  3C and D). A mid-dorsal longitudinal groove extends along the whole length of the dorsal side of the forepart; on the ventral side there is a shallow groove that extends posteriorly from the ends of the bridle keels. The brown bridle keels are always separate, turning posteriorly and running parallel on both the dorsal and ventral side (Fig. 3 B and C).
A slight external groove separates the forepart from the trunk. on the anterior part of the trunk there are two rows of about 40 metamerically arranged papillae separated by a deep and narrow median furrow (Fig. 3B). Each papilla is topped by an oval cuticular plaque, about 70 x 40 μm, with a thickened, bowshaped front edge. Posteriorly, the mid-dorsal furrow widens and another up to 100 papillae, not so tightly arranged, with plaques, continue along its edges. The ventral side of the trunk is slightly concave and latero-ventrally there are numerous small, plaqueless papillae tightly arranged (Fig. 3C). on the ventral side, anterior to the girdles, there is a series of digitiform ridges forming a deep sulcus (Fig. 3F). on the dorsal side there is a group of about 10 enlarged papillae anterior to and between the girdles, and a larger papilla behind the posterior girdle (Fig. 3E).
The two girdles, lying on very thick muscular ridges, are close together, approximately 0.5 mm apart, and are both interrupted dorsally and ventrally ( Fig. 3E and F). The chaetae are arranged in 5 rows,  Additional male characters. The male genital papillae are large and overlap the posterior end of the forepart. The spermatophores are narrow and about 1.75 mm long, with trapezoidal wings and a very long filament (Fig. 3G and 4F).
Distribution. Known only from the type locality, Porto mud volcano, at a depth of 3902 m. Spirobrachia tripeira is the first species of this genus to be described from the Atlantic ocean, so far this genus has only been known from high latitudes, both South and north (Table 2).
Remarks. The genus Spirobrachia was proposed by ivanov (1952) and was recently revised by Smirnov (2000b). in this revision, the species formerly ascribed to Spirobrachia were separated based on the structure of the spermatophore and tube, and Volvobrachia was erected. The spermatophores of Volvobrachia are wide, without clearly differentiated wings, and its tube has a rigid funnel-like mouth, whereas Spirobrachia possesses narrow spermatophores, with a pair of wings at the base of the filament, and the tube has a soft anterior end. The characteristics of the new species described herein are consistent with those of the genus Spirobrachia.
in S. tripeira the arrangement of the bridle is similar to that of S. orkneyensis. The number of tentacles and the presence of tentacular pinnules are also comparable to those of both S. orkneyensis and S. grandis, but S. tripeira differs from these species by the absence of the lophophore. S. leospira, which does not have a lophophore either, can be easily distinguished by having less than half the number of tentacles (Table 2).
DiSCuSSion it was not until 1962 that the first record of frenulates from the western Atlantic was made (Bayer, 1962) and for a long time the number of records from the Atlantic ocean was surprisingly low when compared with that of the Pacific. But with the increased effort put into deep-sea sampling during the 1960s and 1970s, 46 species belonging to 10 genera were described from both sides of the Atlantic (nielsen, 1965;Southward, 1968Southward, , 1971Southward, , 1972Southward, , 1978aSouthward, 1958, 1967). in the following two decades 3 species of Siboglinum from the Eu-   ropean margin were discovered (Flügel and langhof, 1983;Flügel, 1990;Flügel and Callsen-Cencic, 1993), and more recently Smirnov (2000c) described one new species found in the Haakon Mosby mud volcano (off northwestern norway). From the South Atlantic Sector of the Antarctic 7 new species were recently described (Smirnov, 2000a(Smirnov, , 2005a and one redescribed (Smirnov, 2000b). in spite of this increase in the number of species, the knowledge on the distribution of frenulates is still fragmentary, with the latest update for the Atlantic ocean dating from 1979 (Southward, 1979). The current distribution of frenulates in the north Atlantic can be divided into 3 main areas: the northwest Atlantic (from nova Scotia to Florida), the Caribbean and Gulf of Mexico, and the northeast Atlantic along the European coast. in both the southeast and the southwest Atlantic only one species has been recorded (Table 3).
Three genera of Frenulata, Siboglinum, Polybrachia and Oligobrachia were reported by  for the Gulf of Cadiz, but the latter might have been misidentified because their identification was based solely on the tube. it is possible that these specimens belong to the new genus Bobmarleya but their poor state of preservation did not allow their identity to be confirmed.
The three genera reported herein were collected from Carlos ribeiro (Bobmarleya) and Porto (Spirobrachia and Lamellisabella) mud volcanoes. While the megafauna assemblage is diverse on the flanks of the Carlos ribeiro mud volcano, the top, at a depth of 2200 m, is almost devoid of megafauna with the exception of scattered ophiuroids and Bobmarleya gadensis, which inhabits small depressions of the seafloor. Gas hydrates have been recovered from the sediment and methane concentrations measured on the Carlos ribeiro mud volcano are some of the highest from the Gulf of Cadiz. on the other hand, the top of the Porto mud volcano, at a depth of 3900 m, is covered by a continuous field of clumps of 20 to 50 individuals of Spirobrachia tripeira and an undetermined number of adult and juvenile specimens of Polybrachia sp. These clumps are accompanied mainly by the frenulate Lamellisabella denticulata, stalked hexactinellid sponges and crinoids. Many old tubes and sponge stalks are colonised by epifaunal organisms (hydrozoans, actiniarians, cirripeds and other). Mobile fauna (galatheid lobsters, ophiuroids and holothurians), life traces (burrows with star-shaped feeding marks) and scattered Acharax shells were also observed among the tubes.
However, frenulates were also found in all the other mud volcanoes from which biological samples are available. Although not all the species have been identified yet, molecular studies show that there are at least 9 different species belonging to 5 genera (Hilário et al., in prep.), including the new genus Bobmarleya and two genera, Spirobrachia and Polybrachia, not yet known from the nE Atlantic. These observations increase the number of genera present in the nE Atlantic to 8, the same as in the nW Atlantic. This diversity is probably due to the variety of geological and physical settings. Apart from differences in activity and fluid composition Van rensbergen et al., 2005;Hensen et al., 2007;niemann et al., 2006), mud volcanoes situated in the deeper areas of the Gulf of Cadiz are under the influence of deep Atlantic water masses, whereas the shallower mud volcanoes are strongly influenced by the Mediterranean outflow Water (Peliz et al., 2007). This environmental heterogeneity, acting on larvae dispersal and settlement, is likely to lead to high diversity of frenulates. The Gulf of Cadiz is presently the most extensive cold seepage area known on the European margins, ranging between depths of 200 and 4000 m and including over 30 mud volcanoes. it should therefore be considered of utmost importance for the study of distributional patterns of Frenulata.

ACKnoWlEDGEMEnTS
We are deeply indebted to Dr. Eve Southward (Marine Biology Association, Plymouth) for valuable advice and continuous encouragement during the course of this study. We thank the co-chief-scientists of the TTr16 cruise l. Pinheiro (Departamento de Geociências, universidade de Aveiro) and M. ivanov (Moscow State university), and the chief scientist of the MSM01/03 cruise, o. Pfannkuche (iFM-GEoMAr), for the invitation to participate in the cruises. We gratefully thank A. Calado for his help with the scanning electron microscopy. The line drawings were inked by Joana oliveira. This work was supported by the HErMES project, EC contract GoCE-CT-2005-511234. The first author was supported by the FCT grant SFrH/BPD/22383/2005. rEFErEnCES