The gastropod-symbiotic sea anemone genus Isosicyonis Carlgren , 1927 ( Actiniaria : Actiniidae ) : a new species from the Weddell Sea ( Antarctica ) that clarifies the taxonomic position of the genus

a second species of the sea anemone genus Isosicyonis is described and illustrated from 16 specimens collected in the Weddell sea (antarctica) on the Polarstern cruises ant XVII/3, ant XXI/2 and ant XXIII/8. Isosicyonis striata n. sp. is easily distinguishable externally from the other species of the genus Isosicyonis alba by its pattern: white longitudinal stripes on the column, oral disc, and tentacles. It is also distinguished by internal features including the retractor muscles, parietobasilar muscles, marginal sphincter muscles, number of mesenteries, and cnidae. the genus Isosycionis is currently only known from the southern ocean. both species of Isosicyonis live in association with a gastropod, with a single sea anemone occupying almost the whole shell of its gastropod host. the description of this new species, and our re-examination of Isosicyonis alba, resolves the controversial higher taxonomic position of the genus, confirming its placement within the Endomyaria.

equivocal due to the nature of the marginal sphincter (Riemann-zürneck, 1980;fautin, 1984).In the type species of the genus, Isosicyonis alba (studer, 1879), the marginal sphincter has been described as intermediate between meso-endodermal and endodermal, and may vary in development (Carlgren, 1927(Carlgren, , 1949;;Riemann-zürneck, 1980;fautin, 1984).the intermediate nature of the sphincter raises the question of whether Isosycionis belongs to "tribus" Endomyaria or Mesomyaria, or whether it represents a transitional form between them.In turn, this uncertainly raises questions about the origin and significance of the marginal sphincter (Carlgren, 1927(Carlgren, , 1949;;Riemann-zürneck, 1980).In addition, Riemann-zürneck (1980) questioned whether Isosicyonis belonged in actiniidae, pointing out that the marginal sphincter muscles and the cnidom of I. alba did not correspond to those of the family actiniidae; however, she did not suggest any alternative placement.
our description of Isosicyonis striata n. sp., a new species of Isosicyonis from the Weddell sea, and our re-description of I. alba allow us to address these larger taxonomic questions.We re-describe and illustrate I. alba based on newly collected material, and provide a similar treatment of the characters in order to compare the similarities and differences of the two species in the genus.We find that Isosicyonis has an endodermal marginal sphincter, and thus belongs to Endomyaria.We concur with Riemann-zürneck's (1980) observation that the basitrichs of the column are particularly large in Isosicyonis.However, we found that the cnidae of I. striata n. sp. are generally smaller than those of I. alba, thus diminishing the distinctiveness of the cnidom of Isosicyonis as compared to other groups within actiniidae.
MatERIal and MEtHods the material we studied was collected on the R/V Polarstern cruises ant XV/3, ant XVII/3, ant XIX/3, ant XIX/5, ant XXI/2 and ant XXIII/8 to the Weddell sea, antarctic Peninsula, and scotia arc, antarctica, sponsored by the alfred-Wegener-Institut für Polar-und Meeresforschung in bremerhaven, from 1998 to 2007 (table 1).sea anemones were relaxed on board using menthol crystals and photographed alive.small pieces of tissue from selected specimens were preserved in absolute ethanol for DNA analysis, and then the animals were fixed in 10% seawater formalin.fragments of several specimens were dehydrated in butanol (Johansen, 1940), and embedded in paraffin.Histological sections 7-8 µm thick were stained with Ramón y Cajal's triple stain (gabe, 1968).squash preparations of cnidae from preserved material were measured at 1000x magnification with nomarski differential interference contrast optics.the nomenclature for cnidae is from Mariscal (1974) and Östman (2000), with modifications: the nomenclature for basitrichs and microbasic b-mastigophores follows Carlgren (1940) and Mariscal (1974).Cnidae capsules were chosen randomly for measurement.the frequencies given are subjective impressions based on squash preparations.the presence of each type of cnidae in each tissue was confirmed in histological sections of the tissues.
the newly collected material has been deposited in the zoologisches Institut und zoologisches Museum in Hamburg (zMH), the national Museum of natural History, smithsonian Institution in Washington (usMn), and the collection of the research team "biodiversidad y Ecología de Invertebrados Marinos" at the university of seville in spain (bEIM).for the purpose of comparison, we examined the holotype (which is in three pieces) from the swedish natural History Museum (sMnH): Paractis alba studer, 1879.type-56."Gazelle Exp.; o.Patagonien; 60 fathoms; det.studer".also labelled: "Holotype according to fautin, 2002".

REsults
External anatomy (fig.1).Pedal disc wider than column, major axis to 54 mm diameter, extremely thin, concave, conforms to shape of gastropod shell.
Column flattened in oral-aboral axis, smooth, more or less corrugated in preserved specimens, to 30 mm height.Mesenterial insertions visible in most proximal part of column, near limbus.fosse not prominent.
oral disc to 28 mm wide in retracted preserved specimens but tentacles and mouth visible.Mesenterial insertions visible.Internal surface of pedal disc visible through gaping, large, central mouth.tentacles about 80, restricted to margin, apparently in two cycles; all of similar size, delicate and smooth in living and relaxed specimens, to 15 mm length; in preserved specimens to 7.5 mm length, digiform, transversely sulcated, with perforated tip.
Geographic and bathymetric distribution: Isosicyonis alba is an antarctic and sub-antarctic species, probably circumpolar, from shelf and bathyal depths (fig.4). the type locality of I. alba is off the coast of argentina (studer, 1879).It has also been reported from the Chilean coast, antarctic Peninsula, south shetland Islands, south orkney Islands, and the Ross sea, between 100 and 800 meters depth (Ridley, 1882;fautin, 1984;Riemann-zürneck, 1986;zamponi et al., 1998).
We report it for the first time here in the eastern Weddell sea, and expand its known bathymetric range to 84-928 m.External anatomy (fig.5): Pedal disc wider than column, major axis to 23 mm in diameter, thin, concave, conforms to shape of gastropod shell.

Isosicyonis striata
Column relatively tall in oral-aboral axis, smooth, more or less corrugated in preserved and contracted specimens, to 11 mm height.Mesenterial insertions visible in most proximal part of column, near limbus.fosse not prominent.
oral disc to 10 mm diameter in preserved specimens, slightly wider than column in expanded specimens.Mesenterial insertions visible.large, central mouth.tentacles to 48, restricted to margin, in two cycles; all of similar size, delicate and smooth in living and relaxed specimens, to 10 mm long; in colour (fig.5): Column, tentacles, and actinopharynx of living specimens brown.Column with white-yellowish longitudinal stripes.oral disc with white radial stripes that extend onto oral side of tentacles.Preserved specimens pinkish or yellowish.
Geographic and bathymetric distribution: Isosicyonis striata n. sp. is endemic to the antarctic continental shelf (fig.4), having been collected only in the Weddell sea at depths from 158 to 338 m. dIsCussIon

Variability of Isosicyonis alba
the material studied agrees with previous descriptions of Isosicyonis alba (studer, 1879;Carlgren, 1927;Riemann-zürneck, 1980;fautin, 1984).It corresponds in the distribution, types and catego-ries of the cnidae, and in the chromatic patterns with material identified as I. alba by fautin (1984).We concur with fautin (1984) regarding the material studied and described by Riemann-zürneck (1980): the cnidae in the material studied by Riemann-zürneck (1980) are generally smaller than the cnidae of the antarctic material, and the antarctic material includes some categories of cnidae that are absent in Riemann-zürneck's material (table 2). the most significant difference between the two previous accounts is the differences in the categories of cnidae in the mesenterial filaments.Riemann-Zürneck (1980) refers to microbasic p-mastigophores, whereas fautin (1984) refers to microbasic b-mastigophores.Although it is true that the capsules are difficult to differentiate, we agree with Riemann-zürneck that they are microbasic p-mastigophores (fig.3l).We interpret the slight differences in size of cnidae to population-level differences.Riemann-zürneck (1980) studied specimens from sub-antarctic waters off the argentinean coast, whereas fautin (1984) studied material from antarctic waters.
all of the specimens of Isosicyonis alba we studied have two pairs of directive mesenteries.We saw no differentiated siphonoglyphs associated with the directives.the siphonoglyphs were also not clear to Carlgren (1927), who observed only one pair of directives.Riemann-zürneck (1980) and fautin fig. 7. -Isosicyonis striata n. sp.Cnidae: a) basitrich; b) spirocyst; C) basitrich 1; d) basitrich 2; E) basitrich 1; f) basitrich 2; g) basitrich; H) atrich (1984) described I. alba with two pairs of directives and two siphonoglyphs.the anatomy of this species is difficult to interpret and it is possible that there are two weak siphonoglyphs.We found Isosicyonis alba associated with the gastropod identified as Harpovoluta charcoti (lamy, 1910) (K.linse pers.com.).one polyp takes up the whole gastropod shell, leaving the parietal and palatal edges of the shell free.nevertheless, previous descriptions of I. alba identified the gastropod as Provocator corderoi Carcelles, 1947(Riemann-zürneck, 1980) or as Provocator sp.(fautin, 1984).the specimens studied by Riemann-zürneck (1980) are from the sub-antarctic region, off the argentinean coast; similarly, P. corderoi is distributed in subantarctic waters, along the argentinean coast to the falkland Islands.

Isosicyonis striata n. sp.
Isosicyonis striata n. sp. is different from its congener I. alba in its chromatic pattern, cnidae, internal anatomy, and some details of its general morphology.among our specimens of Isosicyonis, we could distinguish three chromatic patterns: column yellowish-pinkish; column and oral disc brownish, with tentacles slightly lighter; column and tentacles brownish with yellow-whitish longitudinal stripes (the stripes are radial in the oral disc).We consider the first two patterns as variants of I. alba and the third one diagnostic of I. striata n. sp.
the distribution and categories of cnidae in Isosicyonis striata n. sp.differ from those of I. alba. the characteristically large basitrichs and spirocysts in the scapus of I. alba are absent in I. striata n. sp. the basitrichs from the tentacles and filaments are smaller in I. striata n. sp.than in I. alba, with size ranges that do not overlap in the case of tentacle basitrichs.the larger category of basitrichs, and the microbasic p-and b-mastigophores of the filaments of I. alba are absent in I. striata n. sp.furthermore, we found a category of nematocyst in the filaments of I. striata n. sp. that does not overlap in size with the microbasic b-mastigophores of the filaments of I. alba.these nematocysts are absent in I. alba.there is no evidence of distinguishable shaft or spines therefore we identified these as atrichs; however, most atrichs are found to be holotrichs (Cutress, 1955;Westfall, 1965).We will refer to these cnidae as atrichs in I. striata n. sp.pending further study.spirocysts, basitrichs, and microbasic p-mastigophores are the most common cnidae in ac-tiniarians (Carlgren, 1949;fautin, 1988).Holotrichs are common in acrorhagi and catch tentacles of actiniids, and also in the tentacles of actinostolids from chemosynthetic habitats (see fautin and Hessler, 1989;lópez-gonzález et al., 2003, 2005;daly and gusmão, 2007;sanamyan and sanamyan, 2007).although they are not typically found in the filaments of actiniarians, this is not the first time they have been reported from these structures (e.g., sanamyan and sanamyan, 2007).Moreover, holotrichs might be inducible, so systematic weight should not be attached to their presence/ absence (fautin, 1988;Edmands and fautin, 1991).thus, we considered the presence (and the relatively high abundance in all examined specimens) of these nematocysts in the filaments of I. striata n. sp.very remarkable, but not sufficient justification to place it in a different genus to I. alba.In our opinion, both species share enough similarities in general morphology, internal anatomy, and ecological habits (see below) to belong to the same genus.
Isosicyonis alba and I. striata n. sp. also differ in their retractor and parietobasilar musculature.In I. alba, these muscles are very small, almost invisible, whereas in I. striata n. sp., they are distinctly developed.the parietobasilar muscle of I. striata n. sp. is differentiated as an independent lamella in the stronger mesenteries (fig.6a, b, C). furthermore, the marginal endodermal sphincter is stronger and more restricted in I. striata n. sp.than in I. alba.
We could only observe clearly one pair of directive mesenteries attached to a single well-differentiated siphonoglyph in Isosicyonis striata n. sp.there are fewer cycles of mesenteries in I. striata n. sp.than in I. alba.However, this could be due to the size of the animal, as all the specimens collected of I. striata n. sp. are smaller than the those of I. alba. the shape of the column is of debatable value as a taxonomic feature because of its variability according to the degree of contraction (shick, 1991); nevertheless, in I. striata n. sp., the column is remarkably longer than in I. alba.
the geographic and bathymetric distributions of both species overlap: Isosicyonis alba has been found mainly off the argentinean coast, the scotia arc, the antarctic Peninsula and the Ross sea, whereas the known distribution of I. striata n. sp. is restricted to antarctic waters of the Weddell sea.However, there is general concern about the real limits of the geographic and bathymetric distribution of newly or recently described species, especially of those from areas such as the southern ocean where sampling effort has been heterogeneous.In antarctica, we have always found I. striata n. sp. and I. alba associated with the gastropod identified as Harpovoluta charcoti (K.linse pers.com.).specimens of I. alba from the argentinean coast are associated with gastropods identified as Provocator corderoi, a species not present in antarctica.

Taxonomic position of the genus Isosicyonis
In his speculative treatment of the origin, development, and evolutionary meaning of the mesogleal and endodermal sphincter, Carlgren (1927) placed Isosicyonis alba within Mesomyaria.However, in 1949 he revised his opinion, placing it instead in Endomyaria.Carlgren (1949) described the sphincter as mesogloeal in small specimens and endodermal in large ones.However, Riemann-zürneck (1980) described the reverse, considering the sphincter mesogloeal in large specimens and endodermal in small ones; she hypothesized that the apparently endodermal nature of the sphincter relates to the shape the anemone adopts when adapting to the gastropod's shell.based on this interpretation, and on the cnidom of I. alba, Riemann-zürneck (1980) argued for removing Isosicyonis from actiniidae, but did not propose any alternative placement for the genus.fautin (1984) concurred with Riemann-zürneck (1980) regarding the nature of the sphincter of I. alba.However, the sphincter muscles of her larger specimens of I. alba do not have such an endodermal appearance as those described by Riemann-zürneck (1980), and she therefore argued for further study of the sphincter before assigning Isosicyonis to a new higher taxon (fautin, 1984).
only the smaller specimens of Isosicyonis alba examined in the present study show a meso-endodermal to endodermal morphology of the sphincter (fig.2C).large specimens show an endodermal but diffuse sphincter (fig. 2d). the endodermal nature of the sphincter does not seem to be due to the effects of the anemones adapting their shape to the gastropod's shell.It seems more probable to us that it is more difficult to observe and interpret this small muscle in histological sections of small individuals.furthermore, the endodermal nature of the sphincter of I. striata n. sp. is clear.Molecular evidence supports the placement of Isosicyonis within actiniidae (unpublished data).
one remarkable characteristic of the cnidom of the two species of Isosicyonis is the large basitrichs of the tentacles and column (especially in the column of I. alba).despite the comments by Riemannzürneck (1980), large basitrichs are common in other taxa currently placed in actiniidae, including the genera Bolocera and Phymactis among others (see fautin, 1984;Häussermann, 2004).nevertheless, the family actiniidae is one of the largest families of actiniaria (Carlgren, 1949), and its monophyly is far from certain (daly et al., 2003); thus, it is probably necessary to subdivide this family.More studies on the phylogeny of actiniaria and on the significance of the cnidom are necessary to address this question.
another feature of the genus Isosicyonis is the symbiotic association of its species with gastropods.Currently, sea anemone species known to live in symbiosis with gastropods or associated with their shells belong to four families: Hormathiidae, sagartiidae, sagartiomorphidae (the three families included in acontiaria), and actiniidae (Endomyaria) (Carlgren, 1949;daly et al., 2004).the relationship between Isosicyonis and the other actiniid symbiotic genus, stylobates dall, 1903, and the relationship of these to the acontiarian symbiotic genera, remains to be investigated further.aCKnoWlEdgEMEnts special thanks are addressed to Prof. Wolf arntz (alfred-Wegener-Institute, bremerhaven) and Josep-Maria gili (Institut de Ciències del Mar, CMIMa-CsIC, barcelona) who made our cooperation and participation in several antarctic cruises possible.
fig. 5. -Isosicyonis striata n. sp.External anatomy: a) lateral view of a semi-expanded living specimen on the host [bEIM(ant-4555)]; b) lateral-oral view of a living specimen with expanded oral disc showing the pattern [bEIM(ant-4944)]; C) lateral view of a preserved specimen on its host [bEIM(ant-4555)]; d) lateral of a preserved specimen on its host [bEIM(ant-4555)].scale bars: a, b, C, 10 mm; d, 20 mm