Lamellibrachia anaximandri, Southward & Andersen & Hourdez, 2011

Southward, Eve C., Andersen, Ann C. & Hourdez, Stéphane, 2011, Lamellibrachia anaximandri n. sp., a new vestimentiferan tubeworm (Annelida) from the Mediterranean, with notes on frenulate tubeworms from the same habitat, Zoosystema 33 (3), pp. 245-279 : 253-272

Lamellibrachia anaximandri n. sp. ( Figs 3-13 )

Lamellibrachia sp. – Woodside et al. 1997: 127. — Olu-Le Roy et al. 2004. — Hughes & Crawford 2006: 1. — Duperron et al. 2009: 395-406.

HOLOTYPE. — Anaximander Mountains , eastern Mediterranean. MEDINAUT, Kazan MV, dive MN12 , sample BT2, 35°26’N, 30°33’30E, 1700 m, 5.XII.1998, Š ( MNHN POLY TYPE 1512 ).

PARATYPES. — Anaximander Mountains , eastern Mediterranean . MEDINAUT, Kazan MV, dive MN12 , sample BT2, 35°26’N, 30°33’30E, 1700 m, 5.XII.1998, 2 specimens ( MNHN POLY TYPE 1513 ; MNHN POLY TYPE 1514 ). — Kazan MV, dive MN12 , sample BT9/10B, 35°26’N, 30°33’30E, 1700 m, 5.XII.1998, 1 specimen ( MNHN POLY TYPE 1515 ). — Faulted Ridge , dive MN9 , sample stn 2, 35°27.3’N, 30°24.5’E, 1300 m, 30.XI.1998, 1Š ( MNHN POLY TYPE 1516 ). — Faulted Ridge , dive MN9 , sample BT8, 35°27.3’N, 30°24.5’E, 1300 m, 30.XI.1998, 1 Š( MNHN POLY TYPE 1517 ).— Amsterdam MV, dive MN 8 , sample BT 1, 35°20’N, 30°16.30’E, 2030 m, 29.XI.1998, 1 ş ( MNHN POLY TYPE 1518 ). — Kazan MV, dive MN12 , sample BT2, 1 specimen ( NHM 2010.233 ). — Kula MV, dive MN11 , sample BT, 35°44’N, 30° 27 30’E, 1630 m, 2.XII.1998, 1 specimen ( NHM 2010.232 ). — Faulted Ridge, dive MN9 , sample BT7, 35°27.3’N, 30°24.5’E, 1300 m, 30.XI.1998, 1Š ( NHM 2010.228 ). — Amsterdam MV, dive MN8 , sample BT1, 35°20’N, 30°16.30’E, 2030 m, 29.XI.1998, 1 specimen ( NHM 2010.231 ) .

Olimpi Field. Eastern Mediterranean, MEDINAUT, Milano MV, dive MN 5, sample BT4, 33°44’N, 24°47’E, 1955 m, 21.XI.1998, part of 1 mature ş (MNHN POLY TYPE 1519). — Milano MV, dive MN 5, sample BT4 33°44’N, 24°47’E, 1955 m, 21.XI.1998, 1 specimen (NHM 2010.230). — Napoli MV, dive MN4 33°43’N, 24°41’E, 1945 m, 22.XI.1998, sample BT, 1 Š (NHM 2010.229).

EMPTY TUBES. — Anaximander Mountains , eastern Mediterranean. Kazan MV, dive MN12 , sample BT9/10B, 35°26'N, 30°33’30E, 1700 m, 5.XII.1998, 2 large empty tubes ( MNHN add type 1512( BT9 ); add type (10B)). Other MEDECO specimens at present being investigated by Eve Southward at Plymouth and Ann Andersen at Roscoff will be returned to CENTOB, IFREMER, Centre de Brest, Plouzané, France .

OTHER MATERIAL EXAMINED. — Nile deep-sea fan. Vestimentiferans collected by the NAUTINIL Expedition during three dives in the Central Province and one dive at Aton East Delta, 9 individuals have been measured (for localities and depths, see Table 1 and Fig. 1 ), these and other Nile fan specimens are to be retained for study at Roscoff. Wreck of S.S. Persia: 110 km SE of Crete, exact co-ordinates not available, depth 2800 m. several dry tube fragments and parts of 3 animals have been examined, in the possession of David Hughes, SMBA, Oban. Anaximander Mountains and the Nile fan: 2007 MEDECO Expedition, Olimpi Field, living specimens were studied ( Table 1).

ETYMOLOGY. — The specific name anaximandri is derived from the region of the first discovery, the Anaximander Mountains, which were named after a Greek geographer of the 6th century B.C.

DESCRIPTION

Tubes

The full-grown tubes from the Anaximander and Olimpi regions have a thick, hard wall and a generally smooth exterior (Fig. 4A, B). They are grayish or brownish white in colour. The maximum diameter measured is 9 mm, but most tubes have a maximum diameter of between 3 and 6 mm, at the anterior end (Fig.4C-F).The middle part of the tube is about 3 to 3.5 mm in diameter, while the posterior part tapers to 2 mm or less, ending in a long, dark brown “root” about 1 mm diameter. The anterior 30 to 50 mm of the tube has a slightly rough surface and transversely striped appearance, produced by short overlaps of the outer layers (Fig. 4D-F), but there are no projecting collars and no anterior funnel (Fig. 4B). Behind this region the surface becomes smoother. A total length of 80 cm was reported by Olu-Le Roy et al. (2004), but during the MEDECO cruise, the longest tube measured was 153cm.However,most of the available tubes are broken. The anterior ends of some tubes have clearly been renewed during life, after suffering damage.

Animals

Live specimens from the Nile Fan and Olimpi region are shown in Figure 5.

Eighteen fixed animals inhabiting the full-grown tubes from the Anaximander and Olimpi regions described above are referred to here as the “ type series” and correspond to fixed specimens illustrated in Figure 6 . The obturaculum diameter ranges in these specimens from 2.7 to 5 mm (mean = 3.5 mm, n = 13) and the length from 5.5 to 12 mm (mean = 8.9, n = 15) ( Figs 6A, B ; 7B ; Table 3). The two halves of the obturaculum form a funnel at the top, extending for about half the length of the plume ( Fig. 6B ). The inner and outer surfaces are smooth. The narrow stalk is elliptical in cross section. The plume has from 3 to 9 pairs of outer sheath lamellae and 8 to 17 pairs of branchial lamellae. The filaments of the sheath lamellae adhere closely together except at their extreme tips and they have no pinnules or bands of cilia. The branchial filaments have two rows of short pinnules and two longitudinal bands of cilia near their tips ( Fig. 6C ), but the proximal region lacks pinnules and cilia.

D-F

FIG. 4.— Tubes of Lamellibrachia anaximandri n. sp. from Anaximander Mountains: A, full-grown tube (dive MN12 ,Kazan mud volcano) ; B, anterior end of same tube; C, young tube with soft wall (type C) (dive MN12 on Kazan mud volcano) ; D -F, young tubes with small collars (type D) (dive MN9 on Faulty Ridge). Scale bars: A, 100 mm ; B, 10 mm; C-F, 5 mm.

The pinnulate region may be damaged or even missing in larger specimens. The obturaculum is stiff and retains its proportions after fixation, but the muscular vestimental region contracts variably. After fixation inside the tube, the diameter of the vestimental region ranges from 2.4 to 4.9 mm (mean = 3.35 mm; n = 17) and the length ranges from 14 to 55 mm (mean = 31.5 mm; n =16). The photographs of living animals from the Nile fan and Napoli MV in Figure 5 confirm the funnel shape and proportions of the obturaculum ( Fig. 5 A-D) and show the length and position of the branchial and sheath lamellae. In fixed specimens the ratio of vestimental length to obturacular length (VL/ OL) varies from 1:1 to 4.9:1 (mean = 3.7; n = 15). ( Fig. 7B, E ). The anterior ends of the vestimental folds form a collar about 1 mm high at the base of the plume ( Fig. 6A, D ). The posterior ends of the folds are separated by a wide ventral gap ( Fig. 6E ). The ventral ciliated field is 1 to 2 mm wide.

Males have a pair of dorsal genital grooves ( Fig.5D, E, live male from Napoli MV) flanked by very narrow epidermal folds, extending from paired gonopores near the hind end of the vestimental region, forward to the collar region. The spermatozoa are elongated, like those of other vestimentiferans and their spiral heads are about 30 µm long. Immature females have no obvious gonopores or grooves,but in a fixed mature female specimen from Napoli MV, lacking a plume and the anterior part of the vestimental region, two very small gonopores are visible on the dorsal side, about 8 mm anterior to the posterior end of the vestimental region.The right pore is a little anterior to the left ( Fig. 6F , arrows). Dorsally, the vestimental folds of this mature female are covered by wide bands of soft epidermis, 250-400 µm in thickness, separated

of another small specimen from the same area as Figure 5F, dive 343, still inside thin-walled tube. Colour photos: Ann Andersen and Matthieu Bruneaux. Scale bars: A, 10 mm; B, 4 cm; C-E, 5 mm; F, G, 1 mm.

C G

H

H, I I

B

)

mm 14

(12

length 10 8

Obturaculum 2 0

4 6

0 10 20 30 40 50 60 Vestimentum length (mm)

C

10

lamellae 8 7

9

6

sheath 5 4

Pairs 2

3

1

0

0 1 2 3 4 5 6 7 8 9 Anterior tube diameter (mm)

E

) 14

mm 12

(

length 10 8

Obturaculum 0 2

4 6

0 10 20 30 40 50 60 Vestimentum length (mm)

F

10

lamellae 8

9 7

6

sheath 5 4

Pairs

3 2

1

0

0 1 2 3 4 5 6 7 8 9 Anterior tube diameter (mm)

by a mid-dorsal strip of thinner, smooth-surfaced epidermis, which divides into two, anterior to the gonopores. The gonopores open onto these paired strips, which are not ciliated.A few oocytes (100 µm diameter)have stuck to the strips, near the gonopores, indicating recent expulsion from the gonoducts.

Cuticular plaques are present on the vestimental and the trunk papillae.The maximum diameter of the oval plaques on the vestimental region ranges from 50 to 70 µm, while those on the anterior part of the trunk are slightly larger, ranging from 60 to 80 µm ( Fig.6H, I ). The trunk is probably as much as 500 mm long

C ch1

in large individuals, but the only complete animal in the type series has a shorter trunk (80 mm) and a regenerating opisthosome 2.5 mm long and 2 mm wide, with11 chaetigerous segments.Long fragments of trunk have been found inside broken tubes.Several isolated opisthosomes have been found in the samples, the largest opisthosome being 7 mm long by 2 mm wide, with about 50 chaetigerous segments.The chaetae are arranged in irregular single to double rows. Their heads are 5 to 7 µm long by 2 µm wide, with an anterior group of 4 to 6 teeth and a posterior group of 4 rows of about 4 teeth each ( Fig. 6G ).

Small specimens

Tubes less than 3 mm in anterior diameter, collected at three sites in the Anaximander region (Amsterdam MV, Kazan MV and Faulted Ridge; Table 1) were examined and the animals inhabiting them were measured. An additional small specimen was collected in the Olimpi Region, at Milano MV. The relationships between various dimensions of small and large specimens can be compared ( Fig. 7 ). There is greater variability in the older population than in the younger. The correlation coefficients R2 are greatly increased by the inclusion of small specimens; compare Figure 7A and D, B and E, C and F .

Juveniles. These are defined here as individuals inhabiting tubes less than 1 mm in diameter and less than 40 mm long. Numerous such young tubes, established on pieces of calcareous crust, were collected at Kazan MV (MN10 BT1); four others were found among a sample of larger tubes from Faulted Ridge (MN9 BT8). Most of them adhere to the substratum for at least 15 mm of their length and have a sealed posterior bulb. The anterior part of the tube lifts away from the rock. There are no collars.

The smallest juvenile is 0.93 mm long ( Figs 8A, B ; 9A ). It has five branchial filaments 0.13 mm long with two rows of pinnules each. It has no obturaculum, but there is a small remnant of the larval feeding organ ( Fig. 8B , si), the siphon or medial process described for young Ridgeia piscesae Jones, 1985 ( Southward 1988; Jones & Gardiner 1988) and Riftia pachyptila Jones, 1981 ( Jones & Gardiner 1988). The next juvenile stage is represented by three individuals about 6 mm long ( Fig. 9B ). They have several pinnulate branchial filaments, arranged in two or more pairs of lamellae. A small obturaculum (diam. 0.4 mm. length 0.5 to 0.8 mm) is present centrally among the branchial filaments. The opisthosoma has about 6 chaetigerous segments. The third juvenile stage, represented by four specimens about 10 mm long ( Fig. 9C, D ), has more branchial filaments, a considerably longer trunk and a wider vestimental region, but the vestimental and opisthosomal lengths have scarcely increased. There are no sheath lamellae.

B

A-C

D

A

A remnant of the siphon is still present ( Fig. 9D , si) and the obturaculum is slightly longer than in the 6 mm specimens. The opisthosome has from 7 to 10 chaetigerous segments. A juvenile taken from the carbonate crust of the north flank of Cheops MV in the western province of the Nile fan, at about 3000 m depth during the MEDECO cruise, is shown in Figure 5F. It has no sheath lamellae. The opisthosome of another juvenile from the same site has at least 10 chaetigerous segments ( Fig. 5G).

Young specimens in thin-walled soft tubes more than 40 mm long. Amsterdam MV ( MN 8 BT4’). Thirteen tubes of diameter 0.6 to 1.5 mm and up to 160 mm long have soft, limp walls and no collars or segmentation. The posterior ends are flattened or broken.These tubes were stuck together, with some sand grains and tube debris attached. Thirteen animals were measured, eight of these

were complete. The obturaculum diameter is 1 mm or less; and the maximum obturaculum length is 3.9 mm; the vestimentum length is 0.2 to 7.8 mm and the trunk length 5.4 to 27 mm. A greater trunk length of 35 mm was measured in an incomplete specimen. Where present, the opisthosoma is 0.8 to 1.8 mm long.

Young specimens in sinuous, thick-walled tubes. Kazan MV ( MN 12 BT 9/10A). Thirty sinuous tubes with thick, “cartilaginous” walls and an irregular surface have a diameter of 2 to 3 mm and are up to 160 mm long. Their posterior ends are missing.Some have an anterior smooth, thin-walled, cylindrical, region a few mm long (Fig. 4C). Twelve animals were measured, all incomplete posteriorly. The obturaculum diameter is 1.4 to 3.0 mm and the length is 3.0 to 7.2 mm. The vestimentum length is 12 to 30 mm and the incomplete trunk up to 100 mm long. No opisthosomes were found.

Young specimens in straight tubes with stiff walls and collars. Faulted Ridge ( MN 9 BT 8). Seventeen straight tubes with thin walls, short segments and narrow collars (Fig. 4D-F). The walls are colourless to pale yellow and semi-transparent. The anterior diameter ranges from 1.8 to 4.8 mm, the maximum length is 140 mm but the posterior end is missing. Eight animals were measured, all incomplete posteriorly. The obturaculum diameter is 1.2 to 3.5 mm, the obturaculum length 3.3 to 7.5 mm. The vestimentum length is 6.3 to 27 mm and the incomplete trunk up to 35 mm. No opisthosomes were found.

Tube development

The secretion of the tube begins at the time of settlement with the formation of a narrow tube with a sealed posterior end and open anterior end, attached to a hard surface (type A). As the animal grows, the open end of the tube is extended and lifts from the substratum, remaining narrow and thin-walled (type B). In some circumstances, secretion of more copious tube material produces a thicker-walled, softer tube (type C). Both the B and C types then grow episodically, adding segments with narrow collars to the open end (type D). The anterior diameter increases gradually, to at least 3 mm and the new segments become shorter, eventually being visible as slight overlaps (see Fig. 4B, E). The tube wall is thickened internally by more layers of secretion and the external collars or overlaps become eroded to leave a fairly smooth tube surface, culminating in the typical hard, tough, tube described above (Fig. 4A). Extension also takes place at the posterior end of the tube, giving rise to the narrow tapering “root” that may extend deep into crevices or soft sediment, carried by the burrowing activity of the opisthosome and filled by the continuously growing trunk region.

Animal development

After settlement and the first formation of a tube the development of the young animal continues with the differentiation and growth of the vestimental, trunk and opisthosomal regions ( Fig. 8 ). A small vestige of the larval feeding organ (siphon) is retained while the earliest (pinnulate) branchial filaments are being formed ( Fig. 8B , si). The obturaculum begins to develop in the centre of the plume of filaments, enclosed between the first few pairs of branchial lamellae. This resembles the development of the plume and obturaculum in Ridgeia piscesae and Riftia pachyptila ( Southward 1988; Jones & Gardiner 1988). Figure 5F shows a living juvenile about 17 mm total length, without sheath lamellae. The characteristic sheath lamellae of Lamellibrachia begin to develop after this, when the vestimental region has reached about 5 mm in length ( Fig. 10 ). The first two sheath lamellae are each composed of a few smooth filaments in a row, and are based on either side of the mid-ventral line, posterior to the branchial lamellae. More filaments are produced at the two margins, so that the lamellae are gradually extended around the sides of the body, towards the dorsal side. The next two sheath lamellae originate behind the first two, again close to the mid-ventral line, and more pairs behind them, so that as the animal grows in diameter, it develops left and right series of sheath lamellae. The filaments grow in length to enclose the branchial lamellae when the animal is retracted inside its tube ( Fig. 6A, B ). This suggests a protective and possibly a sensory function. Photographs and videos of living Lamellibrachia , in situ , taken on the MEDECO expedition show that when the plume is extended the sheath lamellae open like pink petals around the mass of red branchial lamellae. The plumes of living animals, after removal from their tubes are shown in Figure 5. Any respiratory function of the sheath filaments is likely to be poorer than that of the branchial filaments, because the sheath filaments have a much thicker cuticle, they lack pinnules, and the afferent and efferent blood vessels are narrower than those of the branchial filaments ( Van der Land & NØrrevang 1977: pl. 23, fig.109).

Size at maturity

Young specimens lack external sexual characters. In the fixed material, male genital grooves have been noted in ten specimens ranging from 14 to 35 mm vestimental length, in tubes 2 to 7 mm anterior tube diameter. The female gonopores have been seen in 3 specimens, 26, 36 and 45 mm vestimental length, in addition to the damaged specimen described earlier ( Fig. 6F ). These pores are apparently always very small. The development of the gonads has not yet been investigated.

NILE FAN SPECIMENS

( FIGS 5A, C, F, G; 11 ; 12 ; TABLE 1)

Figure 11A shows numerous occupied tubes emerging from a small scarp or fault in the carbonate crust. Figure 11B shows a cluster of younger individuals with red branchial plumes extended, in a sea-floor crevice in the carbonate crust. The younger tubes in both populations have narrow collars at the anterior end ( Fig. 11C ). The specimens collected in 2003 ( Table 1) have tube diameters in the range 3.5 to 6 mm. They are fairly smooth-walled but some have flared anterior ends with slight collars ( Fig. 11D ). The tube colour is grayish. Specimens that were fixed outside their tubes ( Fig. 12A, B, D ) show the obturaculum and plume clearly. Measurements of 6 specimens and photographs of 3 others show that the obturaculum diameter ranges from 1.8 to 6 mm; the obturaculum length from 6 to 17 mm, the vestimentum diameter from 2.2 to 4 mm and the vestimentum length from 12 to 40 mm. There are from 5 to 7 pairs of sheath lamellae and from 8 to 19 pairs of branchial lamellae ( Fig. 12A ). The epidermal plaques on the vestimentum measure 60-70 µm and those on the anterior trunk 70 to 96 µm (measured in one specimen). The dimensions of the animal and its plaques are within the range found in the type material of L. anaximandri n. sp., apart from a slight increase in the maximum number of branchial lamellae, so based on the morphological characteristics it is probable that the Nile fan specimens belong to this species, but the genetic results are necessary to confirm this.

Sections of the trophosome of specimens of Lamellibrachia collected in the centre of the Nile deep-sea fan ( NL 6) show endosymbiotic bacteria of coccoid shape ranging from 1 to 7 µm diameter (mean = 3.12 ± 1.22 µm, n = 262) ( Fig. 12C ). The bacteria lack the internal membranes that are typical of methanotrophs, but show a clear periplasm between an inner cytoplasmic membrane and an outer cell wall, which is typical of Gram-negative bacteria. They also contain small spherical membranebound bodies similar to the sulfur globules of some thiotrophic bacteria. Based on these morphological characteristics we conclude that the endosymbiotic bacteria are probably thiotrophic.

S.S. PERSIA SPECIMENS ( FIG. 13 )

Hughes & Crawford (2006) reported that hundreds of Lamellibrachia tubes and fragments were found in the mailroom of the Persia, ranging up to 105 cm in length and 9 mm in diameter. Most of the tubes were empty. In the eleven dry fragments of tube examined in the present study, the diameter varies from 3.5 to 9 mm. The colour is dark brown and the surface is generally worn, with slight overlaps remaining. The anterior end of one tube is present ( Fig. 13B ). The top 20 mm has shrunk during drying, but it appears to have been smooth and thin-walled. The next part consists a series of about ten closely overlapping layers, followed by a smooth section about 30 mm long and then another series of overlapping layers ( Fig. 13A ).

Parts of three animals have been examined. One has a complete obturaculum, branchial plume and vestimental region ( Fig. 13C ). The obturaculum measures about 8 mm long by 4 mm in diameter, the vestimental region 17 by 3 mm, and the incomplete trunk 55 by 3 mm. There are 3 or 4 pairs of sheath lamellae and at least 15 pairs of branchial lamellae. The distal parts of the branchial filaments have deteriorated and the pinnules have been lost, but some cilia are visible. The filaments of the sheath lamellae have also deteriorated and are separated quite widely distally, though they adhere together proximally. The vestimental plaques range from 60 to 70 µm in diameter and the trunk plaques range from 70 to 95 µm, measured on one specimen. The dimensions of the animal and its plaques are within the range found in the type material of L. anaximandri n. sp., and it is probable that the Persia specimens belong to this species.

MOLECULAR RESULTS

The phylogenetic trees based on the mitochondrial 16S and COI data exhibit a similar topology ( Figs 14; 15). In both trees, there is a very strong support for a lamellibrachiid clade and a distinct escarpiid clade. The COI tree ( Fig. 14) indicates that Lamellibrachia juni , from the Kermadec Arc (South West Pacific), is clearly very different from all other species of Lamellibrachia . No data are currently available in the databases for the mt16S gene for this species. The COI tree also shows that all the other species of Lamellibrachia form a very well supported clade that comprises species from the Atlantic side (Mediterranean and Gulf of Mexico),

I II III

Gulf of Mexico Gulf of Mexico Escarpia spicata / E. laminata L. luymesi / sp. 1 complex L. sp. 2 / E. southwardae complex

and the Pacific side (Western and Eastern). In this phylogeny, L. anaximandri n. sp. forms a cluster with L. luymesi and L. sp. 1 from the deep Gulf of Mexico, with a fairly good confidence (group I in Fig. 14, 0.92 posterior probability and 57% bootstrap value). Within this cluster, however, L. anaximandri n. sp. is clearly apart from the L. luymesi cluster (supported by a 98% bootstrap value and a 1.00 posterior probability). The second deep Gulf of Mexico species (L. sp. 2) also forms a monophyletic cluster (group II in Fig. 14) that is a sister-group to L. luymesi / L. sp. 1. The relationship of the L. luymesi / L. anaximandri n. sp. cluster with other Lamellibrachia remains unclear because of the short internal branches and both tree construction methods do not yield the same branching pattern.The mt16S tree tends to show the same pattern (groups I and II), but the grouping of L. anaximandri n. sp. with either one of these two groups remains unclear ( Fig. 15). All mt16S sequences for L. anaximandri n. sp. form a monophyletic group (98% bootstrap value but posterior probability below0.50), regardless of the area of sampling in the Mediterranean (Nile fan central pockmark, Anaximander Mountains, and Napoli mud volcano in the Olimpi field).

COMPARISON WITH OTHER SPECIES

Some characteristics of Lamellibrachia anaximandri n. sp. can be compared with those of seven other species of Lamellibrachia ( Table 3). Morphology and dimensions alone do not distinguish all the species from one another very satisfactorily. However, the molecular sequence data ( Fig. 14) clearly separate L. anaximandri n. sp. from L. luymesi .

Lamellibrachia columna is the largest of the seven species, having tubes up to 20 mm in diameter, whereas the tubes of the others are generally less than 15 mm wide at the anterior end. The fullgrown tube of L. anaximandri n. sp. resembles those of L. columna and the holotype ( Fig. 16C ) of L. luymesi ( Van der Land & NØrrevang 1977: fig. 124), because it lacks the wide collars seen in L. barhami , L. satsuma , L. victori and the Gulf of Mexico population of L. luymesi . Lamellibrachia juni , a recently described species from the Kermadec Arc, has an unusually thin tube wall, with small collars and a maximum tube diameter of 8 to 12.8 mm.

The diameter and proportions of the various parts of the animals change as they grow: in Lamellibrachia anaximandri n. sp., the diameter of the vestimental region is slightly less than that of the anterior end of the tube, and the diameter of the obturaculum is about the same as that of the vestimental region. The length of the vestimental region is about 5 to 7 times the tube diameter ( Fig. 7A, D ) in both large and small Lamellibrachia anaximandri n. sp. The number of sheath lamellae increases as the vestimental diameter increases, but there is considerable variation among both small and large animals ( Fig. 7C, F ). The range for the species (3 to 9 pairs) is about the same as in Lamellibrachia luymesi , which has a maximum of 8 pairs, but L. satsuma , L. barhami and L. juni have only 4 or 5. Lamellibrachia columna has 8 to 16 pairs of sheath lamellae. The branchial lamellae are often difficult to count accurately because they stick together, but Lamellibrachia anaximandri n. sp. has probably the fewest (8 to 17 pairs; 19 in a Nile fan specimen). Maxima for other species range from 19 to 25 pairs. Lamellibrachia juni has an exceptionally large number, up to 35 pairs. The ratio of vestimentum length to obturaculum length ( VL / OL) has been used in the past to indicate differences between some genera and species of vestimentiferans, ( Jones 1985; Southward et al. 1995; Miura et al. 1997). In Riftia , the obturaculum is about twice the length of the vestimentum, in Ridgeia, OL and VL are more or less equal, in other genera, VL is longer than OL. Table 3 shows the wide variation in this ratio for each species of Lamellibrachia and indicates that it is not very useful in separating species of Lamellibrachia . The size of plaques on the vestimental and trunk papillae ( Fig. 6H,I ; Table 3) can be a useful character. However, because the plaques are collagenous, not chitinous, they shrink considerably when dehydrated. Measurements from electron micrographs are smaller than those made from specimens in aqueous media and are not directly comparable. In the present case, the plaques of Lamellibrachia anaximandri n. sp., L. barhami , L. columna and L. luymesi from the Gulf of Mexico have all been examined in aqueous media (see Materials and methods). Lamellibrachia anaximandri n. sp. has the smallest plaques (55 to

I II III

Gulf of Mexico Gulf of Mexico Escarpia spicata / E. laminata

L. luymesi / sp. 1 complex L. sp. 2 / E. southwardae

complex

80 µm) and the size is consistent among 12 large specimens. Lamellibrachia luymesi from the Gulf of Mexico has vestimental plaques 55 to 60 µm in diameter and trunk plaques 75 to 85 µm, very similar to those of L. anaximandri n. sp. Lamellibrachia columna has slightly larger plaques, particularly on the trunk. Lamellibrachia barhami has the largest plaques of these three (up to 160 µm on the trunk). In conclusion, the new species is closest morphologically to Lamellibrachia luymesi , but its tube lacks the wide and obvious collars of the Gulf of Mexico specimens. However, the tube of the holotype of L. luymesi from Guyana ( Fig. 16C ) is similar to that of L. anaximandri n. sp. Differences between the animals are the slightly shorter vestimental region and the fewer branchial lamellae of L. anaximandri n. sp. The morphological closeness to L. luymesi concurs with the phylogenetic analysis that places L. anaximandri n. sp. with L. luymesi , L. sp. 1, and L. sp. 2 from the deep Gulf of Mexico ( Figs 14; 15). The observed sequence differences between L. anaximandri n. sp. and the other species, however, are greater than typical within-species distances (see Miglietta et al. 2010) and support the new species status of L. anaximandri n. sp. All mt16S sequences for L. anaximandri n. sp. from the three locations sampled in the Mediterranean form a monophyletic cluster, with structure that reflects the geography. There is therefore no molecular evidence that animals from these different areas belong to distinct species. The possibility remains though, that the areas are inhabited by different species that do not differ in either mtCOI or mt16S sequences. This was observed for L. luymesi and L. sp. 1 that inhabit different depth ranges in the Gulf of Mexico (group I in Figs 14 and 15) and differ morphologically ( Miglietta et al. 2010). This situation was also observed for the genus Escarpia (group III in Figs 14 and 15). However, co-occurring species such as L. sp. 1 and L. sp. 2 in the deep Gulf of Mexico usually differ genetically for the molecular markers we sequenced. Interestingly, all the Gulf of Mexico and the Mediterranean species tend to group together. Cordes et al. (2007) have outlined the potential for larval dispersal from east to west and west to east in the equatorial region between the Gulf of Mexico, Caribbean and West African seeps. Olu-Le Roy et al. (2007) similarly showed that there are two amphi-atlantic complexes of species in the genus Bathymodiolus , based on both morphological and molecular data. Unfortunately, only a portion of the nuclear 28S gene (less variable than mt16S and mtCOI) has been sequenced for the Vigo worm ( Williams et al. 1993), preventing any comparisons with our samples.

In the overall phylogeny, L. juni clearly forms a separate cluster from all other species of Lamellibrachia , as reported earlier by Miura & Kojima (2006). The relationship of the Atlantic cluster ( L. luymesi , L. sp. 1, L. sp 2, and L. anaximandri n. sp.) with the other species of Lamellibrachia remains unclear because of the short branch lengths in the cluster excluding L. juni . These short branches are not due to the saturation of the phylogenetic signal ( COI is used to study the phylogeny of all the vestimentiferan tubeworms; see McMullin et al. 2003) and could indicate either a slow-down of evolutionary rates around that time or a rapid radiation of the populations to form the current genetic species. A slowdown of the evolutionary rate could be attributed to the extreme longevity of Lamellibrachia ( Bergquist et al., 2000) and explain the lack of genetic differences between L. sp. 1 and L. luymesi . Although the deep branches are not well supported, the fact that L. luymesi and L. anaximandri n. sp. form a monophyletic cluster, with all other species more basal, suggests that the genus Lamellibrachia most likely originated in the Pacific and later colonized the Atlantic region. Based on these data, however, the actual path from the Pacific to the Atlantic remains unclear. Collections from additional locations such as the Indian Ocean would be necessary to address this question.

NOTES ON FRENULATE POGONOPHORES

Frenulate pogonophores are scarcely known from the Mediterranean and may be unfamiliar to biologists working there. Ivanov (1970) described the hair-like Siboglinum carpinei and noted the presence of other species near Corsica. IFREMER has collected some specimens from just east of the Gibraltar Strait

C

( BALGIM 1984, ECS, unpublished). Outside the Mediterranean, on the European Atlantic continental slope, there are about 20 species in the Bay of Biscay ( Southward 1979) and at least 15 in the Gulf of Cadiz ( Hilário et al. 2010).

The tubes of frenulates are unbranched and almost cylindrical, tapering slightly toward the posterior end. The tube diameter of most species is between 0.1 and 1 mm; the maximum is about 2.5 mm. The adult length ranges from about 100 mm for the smallest to more than 1 m for the largest. Many species have flexible tubes but some are rigid. The animals can move up and down as they secrete their tube, laying down layers from the inside. The tube wall material is fibrous chitin in a protein matrix, providing strength and elasticity. The wall is often composed of narrow dense rings separated by transparent interspaces (Fig. 17 A-C). In some species, short segments are formed as the tube is added to at the anterior end and sometimes these overlap like nested funnels. Tubes may be transparent and colourless or show various shades of yellow, redbrown or even black. The tube characters, including the diameter, are remarkably specific, though some changes occur as they grow and age ( Ivanov 1963; Southward 1969; Southward et al. 2005).

Various fragments of brown tubes, found in sediment samples collected during MEDINAUT dives on mud volcanoes, have been identified as frenulate pogonophores. Some contain animals. These MEDECO specimens at present being investigated by Eve Southward at Plymouth will be returned to CENTOB, IFREMER, Centre de Brest, Plouzané, France.

POGONOPHORA, FRENULATA Webb, 1969 (= Polychaeta, Siboglinidae , frenulates Rouse, 2001) Family SIBOGLINIDAE Caullery, 1914 (revised Ivanov 1963) Genus Siboglinum Caullery, 1914 (revised Ivanov 1963)

TYPE SPECIES. — Siboglinum weberi Caullery, 1944 . Redescribed by Southward (1961).

REMARKS

The genus Siboglinum is characterized by a single anterior tentacle, with or without pinnules and without epidermal glands;the anterior part of the trunk carries two rows of papillae, containing pyriform glands, almost always without adhesive plaques; there are two or three girdles (annuli) of chaetae; the postannular part of the trunk carries ventral papillae, singly or in small groups,opposed by large dorsal glandular shields; the spermatophores are spindle-shaped; the tube is usually ringed, and sometimes also segmented.

Figures

FIG. 3. — In situ photographs of Lamellibrachia anaximandri n. sp. in the Anaximander Mountain region (courtesy of IFREMER): A, at Kazan mud volcano,a gravel and mud situation,with bivalve shells scattered on the surface;the cluster of tubes appears to be growing upward from the sediment; B, at Amsterdam mud volcano, the tubes emerge from crevices between broken rocks.

FIG. 1. — Map of the eastern Mediterranean Basin, showing the position of the Mediterranean Ridge (black lines), the three areas studied (AM, Anaximander Mountains; NF, Nile fan; OF, Olimpi Field) and the approximate position of S.S. Persia shipwreck (Ú).

FIG. 6. — Lamellibrachia anaximandri n. sp., adults: A, anterior end in lateral view, showing the sheath lamellae almost completely hiding the branchial lamellae and the obturaculum, holotype from Nautile dive MN12,Kazan mud volcano; B, ventral half of the plume and obturaculum of another specimen, cut longitudinally to show the depth of the funnel of a specimen from unlabelled MEDINAUT sample;C, part of a branchial filament with pinnules and two bands of cilia,specimen from Nautile dive MN8 Amsterdam mud volcano; D, E, anterior and posterior ends of vestimental region in ventral view, showing the vestimental folds and the two ends of the ciliated field of a specimen from Nautile dive MN8, Amsterdam mud volcano; F, dorsal surface of posterior vestimental region of large female, with vestimental folds spread outward, two arrows mark the gonopores, from Nautile dive MN5 Napoli mud volcano; G, chaetae, in face and side view,from an opisthosome with 43 chaetigers (sample MN10 BT1);H, I, vestimental and abdominal plaques,respectively. Scale bars: A, B, D-F, 1 mm; C, 0.1 mm; G, 10 μm; H, I, 100 μm.

FIG. 7. — Changing dimensions with growth in Lamellibrachia anaximandri n. sp. from Olimpi and Anaximander regions: A-C, type series (individuals having anterior tube diameter>3.5 mm); D-F, individuals of all sizes including type series. A and D, anterior tube diameter related to length of vestimentum; B and E, obturaculum length related to vestimentum length; C and F, number of pairs of sheath lamellae related to anterior tube diameter.

FIG. 8.— The smallest juvenile of Lamellibrachia anaximandri n. sp., 1 mm long, from Kazan MV, dive MN10 BT1: A, B, whole animal, ventral and dorsal;C, opisthosome.Abbreviations:brf, branchial filaments;ch1 toch4, rows of chaetae;op, opisthosome;pl, plaques; s1, s2, s3, first three septa;si, siphon;tr, trunk;vcb, ventral ciliated band; vf, vestimental folds. Note that the first row of chaetae is anterior to the first septum. Scale bars: 100 μm.

FIG. 9. — Stages in the growth of young Lamellibrachia anaximandri n. sp., from dive MN10: A-C, dorsal views of 1.0, 6.0 and 10.5 mm animals; D, ventral view of anterior end of C in more detail. Abbreviation: si, siphon. Scale bars: 1 mm.

FIG. 10.— Number of pairs ofsheath lamellaein young Lamellibrachia anaximandri n. sp. in relation to length of vestimentum.

FIG. 11. — Lamellibrachia anaximandri n. sp. from the Central Area of the Nile deep-sea fan: A, in situ photograph of abundant live animals, in scarp situation (dive MD 336, 1686 m depth) (courtesy of IFREMER); B, in situ photograph of small patch of live animals emerging from hole in calcareous crust (dive MD 338, 1701 m depth) (courtesy of IFREMER); C, detail from Figure 11A, showing the variety of tube diameters and collar arrangements present in this population; D, anterior ends of tubes (dive NL6). Scale bar: 2 cm.

FIG. 12. — Lamellibrachia anaximandri n. sp., specimens from the Nile deep-sea fan (Nautile dive NL14): A, B, D, anterior ends in ventral, right side and dorsal view; C, TEM of bacteria in trophosome; the inner arrow shows the inner cytoplasmic membrane, the outer arrow shows the outer cell wall. Scale bars: A, B, D, 10 mm; C, 1 μm.

FIG. 13. — Lamellibrachia anaximandri n. sp. from S.S. Persia shipwreck: A, various fragments of tube; B, soft anterior region of tube and narrow overlaps; C, anterior end of animal, lateral view. Scale bars: 10 mm.

FIG. 16. — A, tube of the Lamellibrachia sp. (Vigoworm) from the wreck of the “François Vieljeux”; B, tube of Lamellibrachia luymesi Van der Land & Nørrevang, 1975, from Green Canyon 234, Gulf of Mexico, 540 m (Gardiner & Hourdez 2003; specimen #15 provided by Dr S. L. Gardiner); C, tube of the holotype of Lamellibrachia luymesi from Guyana (fig. 124 from Van der Land & Nørrevang 1977, with permission from the Royal Danish Academy of Sciences and Letters). Scale bars: 10 mm.