Morphology and biology of a new Rhynchospio species (Annelida: Spionidae) from the South China Sea, Vietnam, with the review of Rhynchospio taxa

Adults of a new spionid polychaete, Rhynchospio nhatrangi, inhabit sandy tubes on muddy sand intertidal flats in an estuary of Nha Trang Bay, southern Vietnam. The worms are up to 8 mm long and 0.5 mm wide for 55 chaetigers. They are unique among spionids in the changes in the arrangement of hooks during ontogenesis: first development of tridentate hooks in neuropodia from chaetiger 10, later loss of hooks in chaetiger 10, and replacement of tridentate hooks by heavy unidentate hooks in neuropodia of chaetigers 11–14. Adults are simultaneous hermaphrodites having sperm in chaetigers 11–14 and oocytes from chaetiger 15 to 23–33. Spermatozoa are introsperm, about 300 µm long. Oocytes are about 120 µm in diameter, with a thin and smooth envelope. Fertilization and early larval development occur in a hatchery formed by elongated dorsal capillaries on the posterior chaetigers. Larvae escape from the hatchery probably when they have developed four chaetigers and then continue development in seawater, feeding on the plankton. When larvae have grown to 14–15 chaetigers, they likely undergo gradual metamorphosis and settle on the bottom. Metameric nuchal organs, glandular pouches in neuropodia, metanephridial segmental organs, hermaphroditism, sperm, and early larval morphology are described here for the first time for Rhynchospio species. An identification key is provided to eight currently recognized Rhynchospio species.


Introduction
Rhynchospio Hartman, 1936 is a small spionid group comprised of seven species characterized by latero-frontal horns on the prostomium and branchiae beginning from chaetiger 2. It was considered as a genus by some authors (Hartman 1936(Hartman , 1959(Hartman , 1969Day 1967;Fauchald 1977;Blake and Kudenov 1978;Imajima 1991), and as a subgenus of Malacoceros Quatrefages, 1843 by others (Pettibone 1963;Foster 1971). Sikorski (1994) placed Rhynchospio into synonymy with Malacoceros, but Blake (1996) considered Malacoceros and Rhynchospio as a generic pair analogous to Dispio/Scolelepis and Spio/ Microspio in having branchiae beginning from the first/second chaetiger. Reproductive biology and phylogenetic relationships of Rhynchospio species are unknown. In the present paper, Rhynchospio is treated as a genus of Spionidae. Monophyly and relationships of this taxon will be discussed elsewhere.
In a taxonomic survey on polychaetes from the South China Sea, Vietnam, numerous spionid worms were found on an intertidal flat in an estuary of Nha Trang Bay. These worms fit Rhynchospio characteristics but differed from members of all known species of this taxon. Morphology and some characteristics of the reproductive biology of these new worms are described in the present paper.

Materials and methods
Field collections were made in Nha Trang Bay, South China Sea, southern Vietnam in May to July 2006 ( Figure 1A, B). Sediment samples were collected intertidally and in shallow subtidal by digging with a spade and using scuba equipment. Population density was estimated by counting worms in a square 10610 cm. Samples were sieved with a 500 mm sieve in the field and the live worms were sorted under a stereomicroscope in the laboratory. Adults and larvae (found on the dorsal side of mature worms) were relaxed in isotonic magnesium chloride and examined while alive with light microscopes. Larvae were measured to the nearest 10 mm using an ocular micrometer; the precision of measurement of spermatozoa is maximally to the nearest 0.5 mm (2.0 mm for the acrosome and middlepiece). Drawings were prepared with the aid of a camera lucida. The terminology of chaetae (referring to the anterior, posterior, superior, inferior, and alternating groups of chaetae according to their position in parapodia) in the present paper is based on the homology of chaetae in Spionidae suggested by Radashevsky and Fauchald (2000). Patterns of dorsal ciliation (referring to metameric nuchal organs, intersegmental transverse and longitudinal bands of cilia) will be described elsewhere. After examination, adult worms were fixed in 10% formaldehyde solution in seawater, rinsed in fresh water, and transferred to 70% ethanol. Fixed material was deposited in the Institute of Oceanography, Nha Trang, Vietnam (IONT); the Institute of Marine Biology, Vladivostok, Russia  (Ehlers, 1897); 2, Rhynchospio arenincola Hartman, 1936;3, Rhynchospio arenincola asiatica Chlebovitsch, 1959;4, Rhynchospio inflatus (Foster, 1971); 5, Rhynchospio australiana Blake and Kudenov, 1978;6, Rhynchospio glycera Blake and Kudenov, 1978;7, Rhynchospio foliosa Imajima, 1991;8, Rhynchospio tuberculata Imajima, 1991; Figure 10A. 2. Rhynchospio australiana Blake and Kudenov 1978, p 200-202, Figure 15. Perth, West Australia, Australia. 3. Rhynchospio foliosa Imajima 1991, p 14-17, Figures 6, 7. Usujiri Bay of Hokkaido Island, Japan. 4. Rhynchospio glycera Blake and Kudenov 1978, p 199-200, Figure 14

Adult morphology
Up to 8 mm long and 0.5 mm wide for 55 chaetigers. Holotype 7.5 mm long and 0.5 mm wide for 52 chaetigers. Pigmentation absent on body and palps; brownish pigment present on ventral pair of pygidial cirri in life. Prostomium with two latero-frontal lobes, horns anteriorly narrowing (Figure 2A: ho), bearing small peaks with short non-motile cilia. Caruncle low and indistinct, levelled in end of chaetiger 1. Occipital antenna absent. Two pairs of small red eyes situated trapezoidally. Nuchal organs metameric, extending to chaetiger 22, shorter in small individuals ( Figure 4A). External boundaries between chaetigers not discernible but nuchal organs apparently not extending from one chaetiger to another. First pair of metamers on chaetiger 1 as curved ciliary bands on either side of low caruncle; successive metamers as ciliary ovals on posterior half of chaetigers (Figure 2A: no). Palps slender, as long as five to eight chaetigers, with longitudinal groove lined with fine frontal cilia, latero-frontal compound cilia situated along either side of ciliated groove and beating towards the groove, and short transverse bands of agglutinated compound cilia (cirri) regularly arranged along inner latero-frontal side and beating towards distal end of palp.
Chaetiger 1 with capillaries and postchaetal lamellae in both rami. Capillaries of anterior row in noto-and neuropodia of anterior chaetigers with wide limbation and fine granulation on distal part of shaft; capillaries of posterior row thin, without granulation. Posterior notopodia with only capillary chaetae. In mature individuals, dorsal capillaries on posterior chaetigers longest, directed upwards and curved inwards, forming a bounded space above dorsum. Low prechaetal lamellae present in both rami on anterior chaetigers. Postchaetal lamellae on anterior chaetigers elongated, with narrowing tips; lamellae on posterior chaetigers short and rounded.
Sabre chaetae appearing as gradual transformation of inferior capillaries in neuropodia on chaetigers 10-11, 1-2 in a tuft, having narrow limbation and fine granulation on distal part of shaft ( Figure 3A).
Hooks present only in neuropodia. In juveniles, tridentate hooks from chaetigers 10-11, up to four in a series, accompanied by one to three alternating capillaries and by inferior sabre chaetae throughout. Hooks with only outer hood, with main fang and two small upper teeth situated one above another ( Figure 3C); uppermost tooth tiny in hooks in anterior neuropodia, prominent in hooks in posterior neuropodia. Alternating capillaries winged in anterior hook-bearing neuropodia ( Figure 3B), hair-like, alimbate in posterior neuropodia. In mature individuals, falcate unidentate hooks present in neuropodia of chaetigers 11-14, two to three in a series, and tridentate hooks ( Figure 3D) present from chaetiger 15 onwards ( Figure 4C), up to five in a series. Falcate hooks bigger than tridentate hooks, with narrow hood opened distally, without upper teeth above main fang ( Figure 3E); tiny scale resembling upper tooth present on subdistal convex side in some hooks ( Figure 3F).
Branchiae from chaetiger 2 through most parts of the body, absent from 4-13 posterior chaetigers ( Figure 4B), free from notopodial postchaetal lamellae, flattened, with surfaces orientated parallel to body axis, with two rows of short cilia running along inner surface. Afferent and efferent vessels supporting each branchia giving off a looped branchial vessel and not interconnected by capillaries, thus branchiae having smooth appearance.
Nototrochs from chaetiger 1 onwards, both in juveniles and adults composed of two rows of cilia, separated from branchial ciliation by narrow gap. Nototroch on chaetiger 1 interrupted mid-dorsally by caruncle; nototrochs on successive chaetigers as complete transverse rows (Figure 2A: nt). Intersegmental transverse ciliation absent. Intersegmental longitudinal ciliation from chaetiger 2 onwards, as short bands of short cilia on dorsolateral edges of chaetigers, extending from one chaetiger on to anterior part of successive chaetiger (Figure 2A: lc).
Pygidium with one pair of ventral cirri and one pair of dorsal cirri in worms with less than about 40 chaetigers. Larger individuals having pygidium with one pair of ventral cirri and two pairs of thinner dorsal cirri situated one above another (Figures 2C, 4D). Glandular pouches from chaetiger 5, small in the beginning, composed of one to three ampulaceous glandular cells covered by common envelope (Figure 2B: gp). Ventral side of anterior chaetigers smooth, without rosette-like pores. Intersegmental lateral pouches absent.
Digestive tract with ventral buccal bulb extending below oesophagus to almost end of chaetiger 1 (Figure 2B: vb); gizzard-like structure absent. Relationships between length of nuchal organs (in chaetiger numbers, referring to chaetiger with last nuchal metamer) and total number of chaetigers in worm; (B) relationships between distribution of branchiae (in chaetiger numbers, referring to last branchiate chaetiger) and total number of chaetigers in worm; (C) relationships between start of tridentate (arranged throughout) and unidentate (arranged in chaetigers 11-14) hooks (in chaetiger numbers, referring to first hook-bearing chaetiger) and total number of chaetigers in worm; (D) relationships between number of pygidial cirri and total number of chaetigers in worm; (E) relationships between number of male and female fertile chaetigers and total number of chaetigers in worm; (F) relationships between percentage of male and female fertile chaetigers and total number of chaetigers in worm.
Main dorsal blood vessel anteriorly bifurcated, forming one pair of circumoesophageal vessels. One pair of palp vessels arising from circumoesophageal vessels and extending entire length of each palp, ending blindly in palp tip. Main dorsal vessel extending through 10-11 anterior chaetigers and then transforming into intestinal blood sinus enveloping middle and posterior guts. Main ventral blood vessel formed by fusion of circumoesophageal vessels in chaetiger 2 and extending length of body. Heart body absent. Blood red in life.
Metanephridial segmental organs from chaetiger 4 onwards ( Figure 2B: mn), greenish in life, of same morphology in sterile and fertile chaetigers, gradually increasing in size in middle chaetigers and decreasing again in posterior chaetigers, opening to exterior on antero-lateral sides of chaetigers.

Habitat
Rhynchospio nhatrangi inhabits fragile sandy tubes on muddy sand intertidal flats in the estuary of Kai River in Nha Trang Bay ( Figure 1B). The population density reaches 100,000 individuals per 1 m 2 . No worms were found in shallow subtidal despite intensive sampling in different parts of the bay.
Spermato-and spermiogenesis were not resolved and spermatid aggregations were not evident on semi-squashed preparations of worms in life. The spermatozoa were introsperm with an elongated head about 2 mm in diameter, head + middlepiece ca 250 mm long, pointed spiral acrosome 36 mm, nucleus 10 mm, spiral middlepiece 200 mm, and flagellum 35-60 mm long. Acrosome was anteriorly pointed and middlepiece was posteriorly narrowed; nucleus composed of anterior dark part 6 mm long and posterior transparent part 4 mm long ( Figure 5A). The total length of spermatozoa was about 300 mm. No more than 100 spermatozoa were present in each fertile male segment.
Oocytes were about 120 mm in diameter, with thin and smooth envelope, filled with yolky globules. One to four large oocytes were present in the ovary on each side of the female fertile segment; up to eight oocytes developed per segment and 100 oocytes were produced by one individual for a spawning. Individuals brooding larvae on the dorsum had the next generation of vitellogenic oocytes developing in the ovaries.

Larval development
Spawned eggs, embryos, and larvae up to the four-chaetiger stage were held by the elongated capillaries on the dorsal side of the posterior chaetigers of mature individuals. These capillaries formed a hatchery where fertilization and early development occurred. Some tubes with mature worms were maintained in Petri dishes with seawater in the laboratory. The four-chaetiger larvae escaped from the tubes and entered the seawater. In natural conditions, hatched larvae probably continue development in the water column and feed on the plankton. one pair of short ciliary bands present on sides of neurotroch between chaetigers 1 and 2. Ciliated pit on chaetiger 2 accompanied by three ciliated cells, comprised of largest median cell situated posteriorly and bearing long cilia beating towards the pit, and two smaller lateral cells situated on sides of the pit, with short cilia beating antero-laterally. Nototrochs and grasping cilia absent. Gastrotroch present on chaetiger 3, composed of four ciliated cells. Telotroch composed of five large cells bearing long cilia, interrupted dorsally. Pygidium rounded.
Large spindle-shaped glandular cells with striated contents present in anterior part of prostomium. Two pairs of similar cells present inside pygidium; cells extending beyond the surface of distal end of pygidium as short papillae ( Figure 5B, C: gc).
Digestive tract divided by muscular sphincters into oesophagus, midgut and posterior gut. Midgut with narrow lumen, opaque wall having some small lipid globules.
Main blood vessels developed; blood transparent, without pigment. One pair of protonephridia 1-2 mm in diameter and 30-35 mm long present in chaetiger 1, opening to exterior on posterior ventro-lateral sides of chaetiger ( Figure 5C: pn).

Settlement and further morphological modifications
Smallest juvenile of R. nhatrangi found on the intertidal flat in Nha Trang Bay had 15 chaetigers. The 17-chaetiger individuals had fronto-lateral horns on the prostomium, nuchal organs extending to chaetiger 9, branchiae on chaetigers 2-10, tridentate hooks in neuropodia from chaetiger 10, pygidium with two pairs of cirri, metanephridia from chaetiger 4, and glandular pouches from chaetiger 5 onwards. Individuals with more than 40 chaetigers had three pairs of cirri on the pygidium ( Figure 4D). In large individuals, nuchal organs extended to the end of chaetiger 22, and branchiae were present from chaetiger 2 to 47 ( Figure 4A, B).

Etymology
The name of the species refers to its type locality, an intertidal flat in Nha Trang Bay.

Distribution
South China Sea, Vietnam.

Discussion
Analysis of the morphology of different sizes of individuals of R. nhatrangi shows that in late juveniles hooks are lost from chaetiger 10, and tridentate hooks are replaced by heavy unidentate hooks in neuropodia of chaetigers 11-14 ( Figure 4C). Gradual age loss of hooks (actually, replacement of hooks by capillary chaetae) from anterior neuropodia has been reported for many spionid taxa (see Radashevsky and Fauchald 2000) but replacement of one kind of hook by another is only known for Pygospio elegans Claparède, 1863 and Glyphochaeta laudieni Bick, 2006. In P. elegans, bidentate hooded hooks first develop in neuropodia from chaetiger 8 and later are replaced by spoon-like hooded hooks in chaetigers 8-11 (Sö derströ m 1920, p 77;V. I. Radashevsky, unpublished data). In G. laudieni, dentate hooks first develop in neuropodia from chaetiger 8 and later are replaced by heavy grooved hooks in chaetigers 14…20 (Bick 2006). Functional significance of those replacements is unknown. Remarkably, other Pygospio and Rhynchospio taxa have no such replacement and possess one kind of hook in neuropodia throughout life. Glyphochaeta Bick, 2006 is a monotypic taxon, of which the relationships with other spionids are not known.
Rhynchospio nhatrangi is also unique among spionids in the morphology of spermatozoa. Little is known about sperm morphology in Rhynchospio taxa but two other species from the Asian Pacific possess introsperm different from those of R. nhatrangi (V. I. Radashevsky, unpublished data). Perhaps all Rhynchospio members are hermaphrodites and have a similar kind of reproductive biology, including fertilization of thin-envelope oocytes by elongated spermatozoa and brooding of larvae on the dorsum, in a special hatchery formed by elongated dorsal capillaries on posterior chaetigers. Different sperm morphology in the three studied species requires further understanding of their fertilization biology. Limited sperm production (tens of spermatozoa in R. nhatrangi versus tens of thousands of spermatozoa per segment in the majority of other spionids) should be taken into account. It is possible that long spermatozoa are kept more easily by the elongated dorsal capillaries and thus provide higher efficiency of fertilization within the hatchery. Elongated kinds of spermatozoa might have evolved in the common nearest ancestor of Rhynchospio taxa and been maintained by natural selection in all its descendants practising the same reproductive biology. The exact shape of the spermatozoa in those species is an unessential and therefore variable parameter. Self-fertilization may increase efficiency of fertilization unless sexual behaviour and sperm exchange have evolved in these hermaphroditic spionids. What kind of fertilization occurs in reality will be investigated in a further study.
Revising Malacoceros (Rhynchospio) taxa, Foster (1971, p 55) suggested that the species ''have previously been separated primarily on the basis of the anal cirri''. She found that specimens in one sample from Florida had variable numbers of cirri and concluded that this character is not reliable for the specific diagnostic. Based on Ehlers' (1897) and Chlebovitsch's (1959) descriptions, on four syntypes of R. arenincola from California, and also some additional Rhynchospio specimens from the Pacific and Atlantic coasts of the USA, Foster (1971) placed R. arenincola and R. arenincola asiatica into synonymy with M. (R.) glutaeus. Foster (1971, p 56) noticed that ''future collections and examination of material from the type locality of M. glutaeus may demonstrate that they are not synonymous but, at the present, based on available literature and collections, they cannot be distinguished''. Such a synonymization was followed by many subsequent authors but Buzhinskaja (1985) treated the three taxa as different. Taking into account the distance between the type localities of the described Rhynchospio species (Figure 1A), I also doubt the identity of R. arenincola and R. glutaea and suggest they are treated separately (for morphological differences between them, see identification key in the present paper). Rhynchospio arenincola asiatica is here treated as a synonym of R. arenincola but further confirmation of this synonymy is needed. Thus, four Rhynchospio species can currently be distinguished from the northern Pacific, namely R. arenincola, R. foliosa, R. tuberculata, and R. nhatrangi sp. nov.
Morphology and biology of Rhynchospio taxa are poorly known, apomorphic characters have not been revealed, and phylogenetic definition of the taxon has not yet been provided. Two major characters conventionally used to distinguish Rhynchospio taxa, i.e. the presence of latero-frontal horns on the prostomium, and branchiae beginning from chaetiger 2, are homoplasious and also present in other Spionidae members. An identification key for the described Rhynchospio species is given above in the present paper but one must be aware of the uncertainty of some characters in use. Thus, R. australiana and R. foliosa were originally described based on single complete fixed specimens, while R. glycera and R. inflatus were described based on single anterior fragments; original descriptions included a limited set of diagnostic characteristics and neither of these species was redescribed later. Poor information about individual variability, ontogenetic modifications and reproductive characteristics of Rhynchospio species leave their taxonomic position uncertain.
The generic taxa of Spionidae have not yet been defined in terms of their phylogenetic relationships. This is because the earlier analyses of spionid relationships were mainly based on limited sets of characters conventionally used in taxonomic descriptions (Sigvaldadó ttir et al. 1997;Blake and Arnofsky 1999). To succeed in phylogenetic hypotheses, a greater set of characters should be taken into consideration. Among others, those characters should refer to reproductive characteristics, morphology of various ciliary organs, chaetae, digestive, circulatory and excretory systems and their modifications in the course of ontogenesis. A set of those characters will be described elsewhere.