Larval development of Mooreonuphis stigmatis (Treadwell, 1922) (Polychaeta: Onuphidae) from the north-east Pacific

Abstract

Development of Mooreonuphis stigmatis, one of the dominant polychaete species in the intertidal soft-bottom community in False Bay (San Juan Island, Washington, USA) has been studied from the 5-chaetiger stage to adult utilizing SEM techniques. Clearly lecithotrophic larvae develop inside the parental tubes until at least the 17-chaetiger stage. The earliest observed stage demonstrated reduced trochal ciliation. Description of development of various ciliated sensory organs is provided. Development of prostomial and peristomial appendages is similar to that found in other onuphid larvae. The chaetal progression pattern of larvae is described in detail and a unified terminology for larval and adult chaetae is suggested. Three major chaetal progression patterns in onuphids can be recognized. The phylogenetic status of some character states such as pseudocompound falciger dentition, origin of branchiae and subacicular hooks is discussed.

Keywords:

• Chaetal progression
• direct development
• False Bay
• juvenile

Published in collaboration with the University of Bergen and the Institute of Marine Research, Norway, and the Marine Biological Laboratory, University of Copenhagen, Denmark

Introduction

Mooreonuphis stigmatis (Treadwell, 1922) was originally described from the muddy intertidal zone in False Bay, San Juan Island, Washington USA and has since been commonly reported from this area (Hartman 1944, 1968; Banse & Hobson 1974). This species has a wide distribution on the Pacific coast of the USA and Mexico from San Juan Island, Washington to Thurloe Bay, Baja California, and inhabits muddy or sandy environments from intertidal to a depth of 60 m (Hartman 1944, 1968). Although M. stigmatis is very abundant in some soft-bottom communities (Brenchley 1982), there have been no published observations on reproduction or larval development of this species.

All onuphids with known development have lecithotrophic larvae and demonstrate several reproductive strategies classified by Paxton (1993) and divided into four types according to the extent of brood care (Table I). Most species of onuphids belong to type I and have larvae developing in the parental tube until the 20–30-chaetiger stage; in contrast, only a few species have free-swimming lecithotrophic larvae, Paxton's (1993) type IV. Type III sensu Paxton (1993) – development in mucus or jelly masses free or attached to the parental tube – has been reported for four species; type II – development in a mud cocoon – has been described by Day (1960) for one species of Diopatra; however, this observation was considered to be doubtful by Paxton (1993). Paronuphis abyssorum Averincev, 1972, described from the Antarctic, has later been considered to be a junior synonym of Leptoecia vivipara by Orensanz (1990). Both authors found small juveniles developing in the body cavity of adult specimens, so viviparity is one additional type of reproductive strategy present among the onuphids (Paxton 1986, 2005). One more case of viviparity among onuphids has been described by Hartman (1965) for Hyalinoecia bermudensis (Hartman, 1965).

Table I. Summary of different developmental patterns in onuphids (pattern types sensu Paxton (1993) with modifications: I – brooding in the parental tube with direct development; II – viviparity; III – brooding in masses or sacs attached to parental tube; IV – free-spawning with planktonic larvae).

Species	Egg size (µm)	Brood size	Brood care	Type of larva	Type of development	Habitat	Location	Reference
Americonuphis magna (Andrews, 1891)	?	?	Eggs in gelatinous strings	Planktonic, lecithotrophic development	III	Intertidal	North Carolina, USA	Hartman 1945
Brevibrachium maculatum (Estcourt, 1966) as Rhamphobrachium sp.	300–500	10–20	Larvae develop in brood chambers inside the parental tube, egg laying is staggered, 1- to 28-chaetiger larvae found inside one parental tube	Direct development	I	Intertidal	Southern Australia	Smith & Jensz 1968; Paxton 1986
Diopatra albimandibulata Paxton, 1993	?	250	Larvae develop in globular small egg sacs attached to the distal end of parental tube, larvae enclosed by a mucous envelope	Direct development	III	5–18.5 m	Australia, Queensland, Bowen	Paxton 1993
Diopatra? brevicirris Grube, 1856 as D. cuprea Bosc, 1802	?	?	11-chaetiger larvae in the parental tube	Direct development	I		Madeira	Monro 1924; Paxton 1993
Diopatra cuprea Bosc, 1802	220	?	Eggs released in jelly mass which readily dissolved in seawater, larvae reared in the laboratory for 21 days	Planktonic, lecithotrophic development	IV		North East of USA	Allen 1959; Paxton 1993
Diopatra gigova Paxton, 1993	1400	9	Eggs develop in the thin mucous sac on the dorsal side of the worm inside parental tube attached to the tube 1.5 cm from its distal end	? Direct development	I	Intertidal	Western Australia, Dampier Archipelago	Paxton 1993
Diopatra lilliputiana Paxton, 1993	400–900	6–40	Eggs and larvae develop in mucous sac inside parental tube at least to the 15-chaetiger stage	Direct development	I	Intertidal	Western Australia, Broome, Enderby Island, Dampier Archipelago, Noth West cape	Paxton 1993
Diopatra maculata Paxton, 1993	350	1000's	Larvae develop inside mucous matrix which covers the outer distal part of the tube	Direct development	III	Intertidal	Western Australia, Nothern Territory, Queensland	Paxton 1993
Diopatra marocensis Paxton et al., 1995	600	20–100	Larvae develop inside the female tube	Direct development	I	16–40 m	Morocco, Atlantic coast, Sidi Boulbra, fine sand in Abra abra community	Fadlaoui et al. 1995
Diopatra neapolitana Delle Chiaje, 1841	?	?	Eggs develop in the gelatinous mass attached to the distal end of the parental tube	?	III	Intertidal	Italy	Lo Bianco 1898
Diopatra neapolitana Delle Chiaje, 1841	?	?	Artificial fertilization, larvae reared in the laboratory	Planktonic, lecithotrophic development	IV		Arachon, France	Cazaux 1972
Diopatra sp. as D. amboinensis Audouin & Milne-Edwards, 1833	?	?	Up to 6-chaetiger larvae inside the parental tube	Direct development	I		Indonesia as Java	Lieber 1931, cited Paxton 1993
Diopatra sp. as D. sp.nov.	‘Gigantic’	?	Larvae found in the parental tube	Direct development	I		Indonesia as Sumatra	Lieber 1931, cited Paxton 1993
Diopatra sugokai Izuka, 1912 as D. neapolitana Delle Chiaje, 1841	200	?	Eggs in jelly mass attached to the tube opening, artificial fertilization, larvae reared in the laboratory	Planktonic, lecithotrophic development	IV	Intertidal, sandy flat	Japan	Choe 1960
Diopatra tuberculantennata Budaeva & Fauchald, 2008	?	5	Five 28-chaetiger larvae were found in the parental tube	Direct development	I	0–2 m	Caribbean Sea, Belize, west of Dangriga	Budaeva & Fauchald 2008
Diopatra sp.	?	?	6 juveniles (8–15 mm) in mud cocoon	Direct development	? I		South Africa	Day 1960
Diopatra variabilis Southern, 1921	600	30–50	Eggs and larvae of various stages develop in jelly membrane inside parental tube, reared to 15-chaetiger stage in the laboratory	Direct development	I	Shallow brackish water	India	Krishnan 1936; Krishnamoorthi 1963
Fauchaldonuphis paradoxa (Quatrefages, 1866)	Not less than 1200	?2	Two 90-chaetiger larvae about 15 mm in length inside parental tube	Direct development	I	20–72 m	Gulf of Mastatan, Mozambique Channel	Paxton 2005
Hyalinoecia araucana Carrasco, 1983	?	45–69	3–13-chaetiger larvae found inside parental tubes	Direct development	I	600 m	Central Chile, off Lebu	Carrasco 1983
Hyalinoecia bermudensis (Hartman, 1965) as Paronuphis bermudensis	?	3	The anterior half of the body has large ova, and the posterior half contains two well developed embryos	Direct development	II	700–2500 m	Off Bermuda	Hartman 1965
Kinbergonuphis dorsalis (Ehlers, 1897)	?	45	Larvae at least up to 25-chaetiger stage develop in a translucent thin-walled folded bag inside parental tube	Direct development	I	Intertidal	Argentina, Patagonia, Puerto Madryn	Orensanz 1990
Kinbergonuphis notialis (Monro, 1930) as Nothria notialis	?	12–28	Female tube has 6–14 partially isolated external lateral capsules with two ova or developing (up to 24 chaetigers) larvae in each capsule	Direct development	I	210–2900 m	Antarctic	Hartman 1967a, b
Kinbergonuphis simoni (Santos et al., 1982)	370	20–30	Larvae develop up to 20-chaetiger stage in cylindrical membranous chambers inside maternal tube	Direct development	I	Shallow water sandy bay, sorted sediment	Tampa Bay, Florida, USA	Hsieh & Simon 1987
Leptoecia vivipara (Averincev, 1972)	?	7	7–13-chaetiger larvae develop in the body cavity	Direct development	II	437–1180 m	Off Enderby Land and Christensen Coast, Antarctic	Averincev 1972 (as Paronuphis abyssorum); Orensanz 1990
Mooreonuphis jonesi Fauchald, 1982	175–190	50–60	Numerous larvae representing different stages found in a single distinct ten-layerd ovoid chamber inside parental tube	Direct development	I	Shallow subtidal, sandy environments	Off Bermuda	Fauchald 1982b
Mooreonuphis stigmatis (Treadwell, 1922)	400	5–16	Up to 18-chaetiger larvae were found in chambers with very thin walls inside parental tube	Direct development	I	Intertidal	San Juan Island, False Bay, Washington, USA	Present study
Notonuphis antarctica (Monro, 1930) as Paronuphis antarcticus	1170	24	Larvae develop at least up to 24-chaetiger stage inside parental tube	Direct development	I	220–1153 m	Antarctic	Hartman 1967a, b; Averincev 1972
Onuphis elegans (Johnson, 1901) as Nothria elegans	230–235	?	Larvae reared in the laboratory from fertilized egg to 60-chaetiger juvenile	Planktonic, lecithotrophic development	IV	Intertidal –23 m	Dillon Beach, California, USA	Blake 1975

The apparent dominance of onuphid species with non-pelagic development inside the parental tube may be skewed by the fact that most of the larvae were found in adult tubes as a by-product of studies for other purposes. Nevertheless, this type of development is very widely distributed within the family and has been reported for both onuphid subfamilies: Hyalinoeciinae Paxton, 1986 and Onuphinae Kinberg, 1865.

The earliest significant descriptions of larval development in onuphids were of free-spawning species such as Diopatra cuprea (Bosc, 1802) from Woods Hole, Massachusetts (Allen 1959), D. sugokai Izuka, 1912 as D. neapolitana Delle Chaije, 1841 from Japan (Choe 1960), and Onuphis elegans (Johnson, 1901) as Nothria elegans from the coast of California (Blake 1975). Following artificial fertilization, development from early embryos to different larval stages was studied in the laboratory. At roughly the same time, fragmentary data about direct development of larvae inside parental tubes of various onuphids were reported. The most complete studies on direct development of onuphids were carried out for D. variabilis Southern, 1921 from India (Krishnan 1936), Mooreonuphis jonesi Fauchald, 1982b from Bermuda (Fauchald 1982b), Hyalinoecia auraucana Carrasco, 1983 from Central Chile (Carrasco 1983), Kinbergonuphis simoni (Santos et al. 1982) from Florida (Hsieh & Simon 1987) and D. marocensis Paxton et al., 1995 from the coast of Morocco (Fadlaoui et al. 1995). All authors reported the presence of provisional larval chaetae that later in development were replaced by definitive adult chaetae as a main feature of onuphid development. While some of these chaetae are similar in several species, the detailed distributional pattern appears to be species-specific and may be useful in larval taxonomy (Hsieh & Simon 1987). Investigations of the jaw apparatus of onuphid larvae have demonstrated that the replacement of juvenile jaws by adult jaws, at least in onuphids with direct development, may be related to the time when larvae have consumed almost all of the yolk deposited in the egg and are ready to leave the parental tube and start to feed (Fauchald 1982b; Hsieh & Simon 1987).

Chaetal replacement in the development of onuphids was described in detail by Blake (1975), Hsieh & Simon (1987), Orensanz (1990) and Paxton (1996). These authors discussed homology of different types of chaetae in onuphids and eunicids, and a general pattern of chaetal progression in larvae and juveniles of onuphids was suggested.

In the present study we describe the larval development of M. stigmatis and compare our data with known developmental patterns of other onuphids. The terminology that was used by different authors to describe provisional larval chaetae appears to be confusing, and comparison of chaetal distribution in larvae of different species is difficult. We have attempted to unify the terminology used to differentiate the various chaetae in both larval and adult onuphids, with a discussion of the general ontogenetic pattern of chaetal replacement in onuphids. Also based on the current knowledge of onuphid development we discuss the apomorphic and plesiomorphic status of selected characters used in studies of onuphid phylogeny.

Material and methods

Mooreonuphis stigmatis was collected in the intertidal zone in False Bay, San Juan Island in July 2007. Adults with tubes were brought to the laboratory and maintained in flowing seawater for a few days. Larvae of different stages were removed from the tubes under a dissecting microscope and relaxed in 3.5% MgCl₂. For light microscopy analysis larvae were preserved in 4% filtered formalin in seawater with subsequent transfer to 70% ethanol. For scanning electron microscopy (SEM) study we followed the protocol of Zograf & Yushin (2004) with modifications. Larvae relaxed in 3.5% MgCl₂ were fixed in 25% glutaraldehyde in 0.1 M cacodilate buffer (twice per 1 h without rinsing, on ice in the dark). Then the specimens were rinsed 3 times for 20 min each with filtered seawater at room temperature and postfixed in 4% osmium tetroxide in 0.1 M cacodilate buffer (2 h on ice in the dark). After fixation, the larvae were run through an alcohol series, with 30%, 50% and 70% ethanol for 30 min in each step and then in 75%, 95%, 100%, 100% ethanol for 10 min in each step. Specimens were critically point-dried with CO₂ in a Balzers CPD-030 critical point-drier using ethanol as the transition fluid. After the larvae were mounted they were sputter coated with 20–30 nm of gold:palladium alloy 60:40 wt% in a Cressington Scientific 108 auto sputter coater. Samples were imaged on a Leica Stereoscan 440 SEM with lanthanum hexaboride electron source. For SEM study of adults and juveniles of M. stigmatis, worms were fixed in 4% formalin and then transferred to 70% ethanol and dehydrated and coated as described above. Examination of larval jaws was not undertaken due to the low number of specimens.

We followed Hsieh & Simon (1987) in defining young individuals found inside parental tubes as larvae and individuals that have left the parental tube and started to build their own tube but remain immature as juveniles. A total of 28 larvae and juveniles represented five different developmental stages, and 10 adults were used for the morphological study. To study early developmental stages, artificial fertilization was attempted in the laboratory following Blake's (1975) procedure; however, no successfully fertilized eggs were obtained.

Results

Mooreonuphis stigmatis is a dioecious species. Females contain spherical, yellow, yolky eggs up to 300–350 µm in diameter. Segments in the posterior one-third of males contain whitish sperm. Preliminary light microscopy analysis of spermatozoa showed that they have elongated heads with a relatively short flagellum.

Larvae were found in the posterior parts of the tubes (5–16 per tube) in very thin-walled chambers isolated from each other. All larvae from one tube represented the same developmental stage and had the same number of chaetigers.

5-chaetiger larvae

The larvae are about 500 µm long, oblong with indistinct anterior and posterior ends and are full of yolk (Figure 1A, B). The anterior part has a fragmented akrotroch, a transverse row of ciliary patches (Figure 1C, D); a prototroch consisting of a number of ciliary fields surrounding the future prostomium and a metatroch of which only two remnant rows of cilia remained ventrally. Simple red eyes are present on the dorsal side of the future prostomium. No segmentation is visible on the dorsal side of the larva. Six pairs of parapodia have started to develop on the ventral side. The two first pairs have rudiments of both notopodial and neuropodial cirri; subsequent parapodia have only ventral cirral rudiments. Five parapodia have aciculae and the first four parapodia bear one early compound bidentate hooded falciger each. Two anal cirri appear in the posterior region of the larva as small rounded buds.

Figure 1.  Early development of Mooreonuphis stigmatis. (A–D) 5-chaetiger larva: (A) lateral view; (B) frontal view; (C,D) fragment of akrotroch. (E,F) 7-chaetiger larva: (E) lateral view, (F) ventral view. (G,H) 11-chaetiger larva: (G) lateral view; (H) ventral view. ak, akrotroch; dac, dorsal anal cirrus; fl, frontal lip; la, lateral antenna; ma, median antenna; me, metatroch; p, palp; pg, pigidium; pr, prototroch; pro, prostomium; vac, ventral anal cirrus.

7-chaetiger larvae

The larvae are about 620 µm long with a distinct anterior end, a thick oval body divided ventrally into ten segments, and a pygidium with distinct conical dorsal anal cirri and tiny rudimental ventral anal cirri (Figure 1E, F). The prostomium bears five rounded rudiments of palps, lateral and median antennae. All larval trochal ciliation has disappeared. Elongated patches of cilia behind the lateral antennae presumably are the beginnings of the nuchal organs. Eyes are present. A transverse fold appears on the border between the prostomium and future peristomium on the ventral side. Aciculae are present in the first seven parapodia. The parapodia are conical and the first four bear dorsal and ventral cirri. The first parapodium has 2–4 provisional pseudocompound bidentate falcigers; these are replaced over chaetigers 2–6 by provisional transitional compound bidentate falcigers, 2–3 per parapodium. The three anteriormost pairs of parapodia have capillary notochaetae, one per parapodium.

10–11-chaetiger larvae

Larvae are about 700–800 µm long, have all major morphological characters of adults: distinct prostomium and peristomium, segmented body and pygidium (Figure 1G, H). The prostomium has five appendages; both palps and antennae are elongated and have developed single ringed ceratophores at their bases. The frontal part of the prostomium develops into two hemispherical frontal lips. The mouth can open and the muscular proboscis with juvenile jaws can be everted ventrally. A single peristomial ring is well developed and separated from the prostomium; however, peristomial cirri are absent. Palpo- and ceratostyles, rudiments of the frontal lips and anal cirri are covered by scattered patches of long sensory cilia. Nuchal organs are short, developing on the prostomium behind the lateral antennae. Eyes are present. Segmentation is prominent on the ventral side, but still not visible dorsally. The larvae are very yolky and presumably do not feed. All parapodia bear provisional chaetae. The first pair of parapodia has cirriform dorsal and ventral cirri and bears two pseudocompound bidentate falcigers and three pseudocompound bidentate hooded falcigers. Parapodia from the second to sixth–seventh chaetigers have 2–3 transitional compound bidentate falcigers that are replaced by 1–2 compound bidentate hooded hooks in subsequent parapodia. Four to five anterior pairs of parapodia bear capillary notochaetae.

16–18-chaetiger larvae

Larvae are about 1.8 mm long and have the shape of young juveniles (Figure 2A, C). The body is elongate; segmentation is well developed on the ventral side and becoming visible dorsally, especially in anterior and posterior regions of the larva. The prostomium is rounded and bears paired elongate palps and three antennae with short 1–2-ringed ceratophores (Figure 2B, D). Frontal lips are oval and are increasingly separated from the prostomium by a groove. Upper and lower lips are developing ventrally. Nuchal organs are becoming wider and longer. Peristomial cirri are now present. The parapodia resemble the adult shape closely, and have cirriform dorsal cirri, and rounded prechaetal and elongated postchaetal lobes. The first two pairs have cirriform ventral cirri which are replaced by glandular pads in subsequent parapodia. All parapodia have long cilia near the ventral bases of dorsal cirri (Figure 2E, F). These cilia appear in the 10-chaetiger larva, but become increasingly prominent in 16-chaetiger larvae and later stages. Five anterior parapodia have notopodial capillary chaetae. At this stage, adult permanent chaetae start to appear. The first pair of parapodia bears two bidentate provisional pseudocompound falcigers and three permanent tridentate pseudocompound hooded falcigers. From the second to seventh parapodia 2–4 transitional compound bidentate falcigers and 1–2 limbate chaetae are present. Starting from the eighth parapodium, compound falcigers are replaced by 1–3 compound larval bidentate hooded hooks; the first single permanent adult bidentate subacicular hook (SAH) is developed on the ninth parapodium; limbate chaetae are present up to chaetigers 13–14. Branchiae are absent. The upper anal cirri become elongate, covered by patches of cilia, ventral anal cirri remain very short and conical.

Figure 2.  16-chaetiger larva of Mooreonuphis stigmatis: (A) dorsal view; (B) prostomium, dorsal view; (C) lateral view; (D) median antenna; (E) parapodium of the third chaetiger; (F) dorsal cirrus of the third parapodium. cil, ciliae; dac, dorsal anal cirrus; dc, dorsal cirrus; la, lateral antenna; ma, median antenna; n, notochaeta, ng, nuchal groove, p, palp; pc, peristomial cirrus; pso, parapodial sensory organ; vac, ventral anal cirrus; vc, ventral cirrus.

22–24-chaetiger juveniles

Juveniles about 2.5 mm long leave the parental tube and start to build their own tubes. This process was observed in the laboratory. Young individuals have a well-developed prostomium and peristomium with all appendages and sensory organs. The yolk is almost consumed and dorsal segmentation is visible along the whole length of the worm except in a few median segments. Ventral cirri are cirriform in the first two pairs of parapodia and are replaced by glandular pads posteriorly. A digitiform postchaetal lobe is prominent in the first 3–4 parapodia. Provisional capillary notochaetae are present only on the first three chaetigers. The first pair of parapodia bears four larval bidentate pseudocompound hooded falcigers, the second pair has four larval bidentate hooded falcigers and four adult tridentate hooded falcigers. Parapodia 3–8 have three provisional transitional compound bidentate falcigers and 2–3 simple limbate chaetae. Starting from the ninth parapodium, 1–2 bidentate larval subacicular compound bidentate hooded hooks appear in each parapodium. At the same time, one adult subacicular simple bidentate hook per parapodium is present on chaetigers 9–17. Pectinate chaetae are present on chaetigers 12–17, one per parapodium. Branchiae are absent.

118-chaetiger juveniles

The juvenile is 32 mm long and 0.9 mm wide (without parapodia) and has all characters of adult individuals but remains sexually immature (Figure 3A). The prostomium is rounded, the two palps reach chaetiger 2, the two lateral antennae reach chaetiger 5 and the median antenna reaches chaetiger 4. The ceratophores have up to four rings (Figure 3C). The frontal lips are ovate and directed forward, the lower lip has a median section (Figure 3D). Eyes are present near the base of lateral antennae. Nuchal grooves have become straight, and very long, narrowly separated from each other. No provisional larval chaetae are present, the chaetal distribution is the same as in adults. The first three pairs of parapodia bear only neurochaetae including 1–2 simple capillary chaetae and 4–5 tridentate pseudocompound falcigers with short blunt hoods. Notochaetae are present inside the dorsal cirri. The fourth parapodium has one tridentate pseudocompound hooded falciger, one large median simple tridentate hooded hook, 10–12 simple limbate chaetae and two compound spinigers. Only limbate chaetae and compound spinigers are present on chaetigers 5–16. The compound spinigers are replaced by simple bidentate subacicular hooded hooks starting from chaetiger 16. Ventral cirri are cirriform on the first four parapodia; the postchaetal lobe remains digitiform on the first 15 chaetigers. Branchiae are simple and strap-like, starting from chaetiger 17 and continuing almost to the end of the worm.

Figure 3.  118-chaetiger juvenile of Mooreonuphis stigmatis: (A) lateral view; (B) parapodial sensory organ of the fifth parapodium; (C) anterior end, dorsal view; (D) anterior end, ventral view. dc, dorsal cirus; fl, frontal lip; la, lateral antenna; ll, lower lip; ma, median antenna; ng, nuchal groove; p, palp; pc, peristomial cirrus; psl, postchaetal lobe; pso, parapodial sensory organ; ul, upper lip; vc, ventral cirrus.

Discussion

Mooreonuphis stigmatis broods embryos and larvae in the parental tube until the 17-chaetiger stage and thus belongs to type I sensu Paxton (1993). All larvae in a single tube are in the same developmental stage and probably are the result of a single spawning event. Presumably several such events can occur during the reproductive cycle.

The mode of fertilization remains unclear. Sperm transfer system involving mushroom-shaped spermatophores containing elongate spermatozoa has been described for Kinbergonuphis simoni (Hsieh & Simon 1990). Although the spermatophores were not observed in M. stigmatis, the elongate shape of spermatozoids suggests that internal insemination may occur in this species; this could in part explain our lack of success with artificial fertilization.

Distribution of cilia in early embryos

Distribution of cilia in early developmental stages has been described for only two onuphid larvae. Pelagic larvae of Onuphis elegans demonstrate complex ciliation that includes an apical tuft, akrotroch, prototroch, telotroch and transverse ciliary bands on all segments at the 5-chaetiger stage. Although ventral ciliation at the posterior margin of the peristomium was described, the term metatroch was not used (Blake 1975). Larvae of Kinbergonuphis simoni have direct development in the parental tube and show less well-developed ciliation. Rudiments of all ciliary bands except the segmental transverse bands are still present, but have patchy structure and never form complete bands. These larvae are unable to swim and stay within the parental tube (Hsieh & Simon 1987). In Mooreonuphis stigmatis, distribution of cilia was also patchy. Although we did not observe early embryos and thus could not trace a reduction of the apical tuft and certain patches of the prototroch, the anterior ciliation of 5-chaetiger larvae of M. stigmatis is almost identical to that of 3–4-chaetiger larvae of K. simoni (Figure 1A–D). No ciliation was observed in the study of 3–13-chaetiger larvae of H. araucana; while cilia may occur at earlier stages of development, it appears probable that the embryos and larvae of this species have completely lost ciliation (Carrasco 1983).

Development of prostomium and peristomium

The first rudiments of prostomial appendages appear at the 5-chaetiger stage as five very small, almost indistinguishable bulges between the rudiments of the akrotroch and prototroch (Figure 1A, B). Later, at the 7-chaetiger stage, all appendages become more prominent. They go from being knob-shaped, through being conical to elongated, approximating the shape of the adult anterior appendages (Figure 1E, F). The first ring of ceratophores appears at the 11-chaetiger stage, the number of rings increases to two in larvae and later to 4–5 in juveniles and adults. Frontal lips first appear at the 10-chaetiger stage as hemispherical buds, becoming oval and finally tapering during later development (Figure 1G, H). Upper and lower lips form later becoming well separated in the 16–17-chaetiger larvae. Peristomial cirri appear very late in development after all prostomial appendages have formed (Figure 2A, B).

Generally, this pattern of development of the anterior region is quite similar in the majority of onuphid species investigated. Kinbergonuphis simoni has the same sequence of formation in all structures, but they form slightly earlier in development: the first signs of prostomial appendages appear at the 3-chaetiger stage; and the late-appearing peristomial cirri become visible at the 7-chaetiger stage. The development of Hyalinoecia araucana is noticeably different. In this species the first three prostomial antennae appear at the 8-chaetiger stage. Palps develop much later, after at least 13-chaetiger larvae. Peristomial cirri are never present even in adults of Hyalinoecia, and do not appear during larval development (Carrasco 1983). Absence of peristomial cirri in adults of some onuphid genera such as Hyalinoecia Malmgren, 1866, Epidiopatra Augener, 1918 and Notonuphis Kucheruk, 1978 is here considered a paedomorphic character.

Development of ciliated sensory organs

The body of all onuphids is covered by scattered small patches of short cilia with the highest density being present on the prostomial appendages, frontal, upper and ventral lips, ventral and anal cirri. According to Pflugfelder (1929) and Paxton (1986), these patches combine sensory and secretory structures. Purschke (2005) defined them as multiciliate penetrative sensory cells. In the larval development of Mooreonuphis stigmatis, the first ciliated structures of this kind appear at the 10–11-chaetiger stage and are represented by small patches of cilia randomly distributed on the body surface, with higher concentration on the prostomial appendages and on dorsal, ventral and anal cirri. Later in development the number of the patches increases, they become denser especially on the anterior part of the body. Kinbergonuphis simoni displays a similar pattern (Hsieh & Simon 1987).

Two patches of relatively long cilia in front of the lateral antennae and four patches behind the lateral antennae are present at the 7-chaetiger stage of M. stigmatis. One pair of these patches is in the same position and has the same shape as the nuchal organs of the next stage. Later in development all long cilia disappear except in these two patches, which become wider and longer and in adults have the shape of very long transverse grooves narrowly separated from each other. Åkesson (1967) stated that in eunicids the trochoblasts do not contribute to the nuchal organs which are developed exclusively from epishere cells. Purschke (2005) agreed with Åkesson's observation; however, he pointed out that no ultrastructure studies on nuchal organs development in polychaetes have been undertaken.

Another sensory organ located at the bases of the dorsal cirri was described by Hayashi & Yamane (1994) for 14 species from different eunicean families including the onuphid, Kinbergonuphis fragilis (Kinberg, 1865). Similar lateral organs have been found in various polychaete families and considered to be homologous (Rouse & Pleijel 2001; Purschke 2005). We found lateral organs in larvae, juveniles and adults of M. stigmatis (Figure 2E, F and 3B). Starting from the 10–11-chaetiger stage, each parapodium has a few long cilia emerging at the base of the knob-like dorsal cirrus. Later in development, cilia increase in number and in adults these sensory organs are of ovoid shape and consist of numerous long cilia.

Development of chaetae

The chaetal progression in onuphids is characterized by the presence of provisional chaetae which occur only in larvae and early juveniles and which are always replaced by the permanent adult chaetae. The number, morphology and distribution of provisional chaetae have been described in detail by several authors (Table II). Description and figures of juvenile chaetae are reported for five species of onuphids (Blake 1975; Carrasco 1983; Hsieh & Simon 1987; Fadlaoui et al. 1995; Paxton 1996) and one eunicid (Åkesson 1967). Orensanz (1990) discussed the homology of different chaetae and provided a comparison of the various chaetal types described by different authors, but did not suggest a uniform descriptive terminology and coding. Paxton (1996) agreed with the homology of provisional and adult chaetae in onuphids as proposed by Orensanz in all but one case. She provided the pattern of chaetal progression in juveniles of Hirsutonuphis Paxton, 1986 and for onuphids in general.

Table II. Different types of provisional larval (lowercase) and permanent adult (uppercase) chaetae in comparison with terminology proposed by other authors.

Species			Mooreonuphis stigmatis		Kinbergonuphis simoni		Onuphis elegans		Hyalinoecia araucana		Hirsutonuphis macrocerata
Type of chaeta			Name	Code	Name	Code	Name	Code	Name	Code	Name	Code
Notopodia			Notopodial capillary chaeta	n	Simple capillary chaeta	n	Simple capillary chaeta	d	Larval simple capillary chaeta	L	–	–
Neuropodia	Falcigers	Anterior modified parapodia	Anterior pseudocompound bidentate falciger	a	Pseudocompound bidentate and unidentate falcigers	a, b	–	–	–	–	–	–
			Anterior early compound bidentate hooded falciger	b^1	–	–	–	–	–	–	–	–
			Anterior pseudocompound bidentate hooded falciger	b^2	–	–	–	–	–	–	–	–
			Anterior compound bidentate hooded falciger	b^3	Compound bidentate hooded hooks	e	Larval compound falcigers limited to first two chaetigers	a^1, a^2	Larval compound falcigers	G, H, I	Composite bidentate falciger	–
			Anterior pseudocompound tridentate hooded falciger	B	Pseudocompound bidentate hooded hook	B	Adult compound falcigers limited to first three chaetigers	a^3	–	–	–	–
		Transitional parapodia	Transitional compound bidentate falciger	c	Compound bidentate falciger	c	Larval compound falcigers limited to chaetigers 2-6	b	Larval compound falciger	?F	Compound median and ventral anterior provisional subacicular hooks	m.a.p.SAH v.a.p.SAH
	Hooks	Posterior parapodia with subacicular hooks (SAH)	Posterior compound bidentate subacicular hooded hook	d	Compound bidentate hooded hook	d	Larval compound falciger limited to chaetiger 5 and successive chaetigers	c	Larval compound falciger	J, K	Posterior provisional subacicular hook	p.p.SAH
			Posterior simple subacicular bidentate hooded hook	D	Subacicular bidentate hooded hook	D	Subacicular hook	f	–	–	Permanent subacicular hook	perm.SAH
	Other chaetae		Large median tridentate hooded hook	G	–	–	–	–	–	–	–	–
			Acicular chaeta	A	Acicular chaeta	A	–	–	–	–	–	–
			Simple limbate chaeta	C	Simple limbate chaeta	C	Simple unilimbate capillary chaeta	e	Larval bilimbate capillary chaeta	M	–	–
			Pectinate chaeta	E	–	–	–	–	Larval comb chaeta	N	–	–
			compound spiniger	F	–	–	–	–	–	–	–	–
Reference			Present study		Hsieh & Simon 1987		Blake 1975		Carrasco 1983		Paxton 1996

We found the coding of different types of chaetae using letters to be very convenient, especially in comparing chaetal distribution of different species. Here we compare different ways of determination and coding of juvenile and adult chaetae of five species of onuphids (Table II) and propose a unified terminology for the different chaetae. This unified terminology is modified from Hsieh & Simon (1987) who provided the most detailed classification of the juvenile chaetae so far published. Some chaetal types are uniform for different genera, others appear to be unique. We use the term ‘falciger’ to cover both provisional and permanent chaetae that occur on modified anterior parapodia of larvae, juveniles and adults and on transitional parapodia of larvae and juveniles. The term ‘hook’ is used for both larvae and adults but only for the subacicular (in Onuphinae) or intrafascicular (in Hyalinoeciinae) chaetae that emerge in posterior parapodia in a ventral position in the fascicle.

Following Hsieh & Simon (1987), we used uppercase letters to code permanent adult chaetae and lowercase letters to code provisional larval chaetae. We used a(b) with a superscript to code various anterior compound or pseudocompound provisional falcigers without (with) hoods. B was chosen to code all permanent adult pseudocompound hooded falcigers. To code transitional falcigers, we used c. For subacicular hooks coding d (for provisional chaetae) and D (for permanent chaetae) were used. All other permanent chaetae were coded with subsequent uppercase letters. n was chosen to code provisional notopodial chaetae and A to code aciculae.

Chaetal composition in Mooreonuphis stigmatis

Larval (provisional) chaetae (Figure 4): notopodial capillary chaeta (n), anterior pseudocompound bidentate falciger (a), anterior early compound bidentate hooded falciger (b ¹ ), anterior pseudocompound bidentate hooded falciger (b ² ), anterior compound bidentate hooded falciger (b ³ ), transitional compound bidentate falciger (c), posterior compound bidentate subacicular hooded hook (d).

Figure 4.  Chaetae found on different larvae of Mooreonuphis stigmatis: (A) anterior early compound bidentate hooded falciger – b ¹ ; (B) anterior pseudocompound bidentate falciger – a; (C) anterior pseudocompound bidentate hooded falciger – b ² ; (D) anterior pseudocompound tridentate hooded falciger – B; (E) anterior compound bidentate hooded falciger – b ³ and transitional compound bidentate falciger – c; (F,G) transitional compound bidentate falciger – c; (H) posterior compound bidentate subacicular hooded hook – d; (I) posterior compound bidentate subacicular hooded hook – d, simple limbate chaeta – C and posterior simple subacicular bidentate hooded hook – D. Lowercase letters represent provisional larval chaetae, uppercase letters represent permanent adult chaetae.

Adult (permanent) chaetae (Figure 5): acicula (A), anterior pseudocompound tridentate hooded falciger (B), simple limbate chaeta (C), subacicular simple bidentate hooded hook (D), pectinate chaeta (E), compound spiniger (F), large median tridentate hooded hook (G).

Figure 5.  Chaetae present on adults of Mooreonuphis stigmatis: (A) pseudocompound tridentate hooded falciger – B; (B) compound spiniger – F; (C) large median tridentate hooded hook – D; (D) pectinate chaeta – E.

Chaetal composition and distribution in M. stigmatis (Table III) appears to be very similar to the distribution in Onuphis elegans (Blake 1975), Kinbergonuphis simoni (Hsieh & Simon 1987), and three species of Hirsutonuphis (Paxton 1996). This pattern was described by Orensanz (1990) and Paxton (1996). Provisional falcigers appear on anterior modified parapodia starting from chaetiger 1 appearing consecutively and are later gradually replaced by permanent falcigers. As each anterior parapodium becomes modified during development, the first event is the appearance of provisional pseudocompound falciger as reported in K. simoni (Hsieh & Simon 1987), H. armillata Paxton, 1996, H. macrocerata Paxton, 1996, H. intermedia Paxton, 1996 , Rhamphobrachium diversosetosum Monro, 1937 (Paxton 1996) and M. stigmatis. These chaetae were considered by Paxton to be homologues to eunicid falcigers, although Orensanz (1990) considered them to be homologous to subacicular hooks. Paxton suggested that the bidentate condition of pseudocompound falcigers was ancestral because such falcigers always appeared first and were later replaced by tridentate in some species. Our observations supported this hypothesis. All anterior provisional falcigers in M. stigmatis were bidentate, while in adults only tridentate pseudocompound falcigers were present.

Table III. The distribution of different chaetae during the development of Mooreonuphis stigmatis. Lowercase letters represent provisional larval chaetae (a – anterior pseudocompound bidentate falciger, b ¹ – anterior early compound bidentate hooded falciger, b ² – anterior pseudocompound bidentate hooded falciger, b ³ – anterior compound bidentate hooded falciger, c – transitional compound bidentate falciger, d – posterior compound bidentate subacicular hooded hook, n – notopodial capillary chaeta). Uppercase letters represent permanent adult chaetae (A – acicula, B – anterior pseudocompound tridentate hooded falciger, C – simple limbate chaeta, D – posterior simple subacicular bidentate hooded hook, E – pectinate chaeta). Numbers represent the number of each chaeta on the parapodium of corresponding chaetiger.

	Chaetiger
Stage	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22
5-chaetiger	1b^1	1b^1	1b^1	1b^1
	1A	1A	1A	1A	1A
7-chaetiger	1n	1n	1n
	2a	3c	3c	3c	2c	2c
	1A	1A	1A	1A	1A	1A	1A
10-chaetiger	1n	1n	1n	1n
	2a	3c	3c	3c	3c	3c	1c
	3b^2						2d	1d	1d
	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A
11-chaetiger	1n	1n	1n	1n	1n
	2a	3c	3c	3c	3c	3c
	3b^2						3d	3d	2d
	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A
16-chaetiger	1n	1n	1n	1n	1n
	2a	3c	3c	3c	3c	3c	3c	2c
	3B	1b^3						1d	2d	2d	3d	2d	2d	2d
		2C	2C	2C	1C	1C	1C	1C	2C	2C	2C	2C	1C
									1D
	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A
22-chaetiger	1n	1n	1n
	4b^3	4b^3	3c	3c	3c	3c	3c	3c
		3B							2d	1d	1d	1d	1d	1d	1d	1d	1d	2d	2d	2d	1d
			2C	3C	3C	2C	2C	2C	2C	2C	2C	2C	2C	2C	2C	2C	2C	1C	1C
									1D	1D	1D	1D	1D	1D	1D	1D	1D
												1E	1E	1E	1E	1E	1E
	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A	1A

Although in general the chaetal progression in M. stigmatis was very similar to that in some other onuphids, one remarkable exception was observed. Usually the first precursors of pseudocompound anterior falcigers appear on the first chaetiger and form successively as development progresses. In M. stigmatis, the four first chaetigers had a single early bidentate compound hooded falciger (b ¹ ) at the 5-chaetiger stage (Figure 4A). At the 7-chaetiger stage, these falcigers had completely disappeared and the usual chaetal progression occurred. Hsieh & Simon (1987) demonstrated that although different onuphid species have similar types of provisional chaetae, the larval chaetal pattern is species-specific. The presence of anterior early bidentate compound hooded falcigers, a unique type of chaeta, may serve as a diagnostic character for M. stigmatis and possibly for genus Mooreonuphis.

We agree with Orensanz (1990) and Paxton (1996) that the provisional compound subacicular hooded hooks are the precursors of permanent simple bidentate subacicular hooks (SAH). Generally, their distribution and location in the parapodia corresponds to the position of permanent hooks in all studied species, but in all cases permanent hooks start to appear a few chaetigers posterior to the first origin of provisional compound hooks. Distribution of permanent hooks shows great variability in adults of different species of onuphids, but in larval development all species demonstrate remarkable similarity in the place of first origin of permanent SAH (Table IV).

Table IV. A comparison of the subacicular hooks (SAH) development among ten species of onuphids.

Species	Stage when first SAH appear	No. chaetiger with the first SAH in juveniles	No. chaetiger with the first SAH in adults	Reference
Diopatra lilliputiana	15-chaetiger	6	14 (10–12)	Paxton 1993
Diopatra marocensis	12-chaetiger	9	13–15	Fadlaoui et al. 1995
Fauchaldonuphis paradoxa	90-chaetiger	9	15–17	Paxton 2005
Hirsutonuphis armillata	?	11	11–12	Paxton 1996
Hirsutonuphis intermedia	?	10–11	13–15	Paxton 1996
Hirsutonuphis macrocerata	?	10–11	15–19	Paxton 1996
Kinbergonuphis simoni	17-chaetiger	9	17	Hsieh & Simon 1987
Mooreonuphis stigmatis	16-chaetiger	9	16	Present study
Onuphis elegans	14-chaetiger	8	9	Blake 1975
Rhamphobrachium ehlersi	?	7–8	15–16	Paxton 1986

Paxton (1986) considered that the early origin (chaetiger 8–19) of the SAH in onuphids was a derived neotenic character compared to the late origin of these chaetae (after chaetiger 20) in eunicids. Comparison of eight species of onuphids demonstrates that all of them have the first permanent SAH appearing on chaetigers 7–11. Later in development some species, such as O. elegans and H. armillata, retain the same condition in the adults, whereas in other species the SAH starts more posterior. In development of Eunice valens (Chamberlin, 1919) the first permanent SAH appears very early, on the chaetiger 6 at the 6-chaetiger stage; however, in adults it may start as late as at chaetiger 43. Here we consider the early origin (before chaetiger 11) of the SAH to be ancestral and late origin (after chaetiger 11) to be derived.

The chaetal progression mode appears to have a high phylogenetic value. Phylogeny of onuphids at generic level proposed by Paxton (1986) had reveled two monophyletic subfamilies: Hyalinoeciinae and Onuphinae. Both subfamilies contain two groups of genera: the Nothria group and the Hyalinoecia group are included into Hyalinoeciinae, and the Diopatra group and the Onuphis group form Onuphinae. The relations between the two subfamilies remain unresolved and the most ancestral genus is unknown (Paxton 1986). Three different modes of chaetal progression are found to be characteristic for three monophyletic groups of genera. Nevertheless, details of provisional chaetal morphology and replacement have been reported only for few species and all phylogenetic interpretations are preliminary.

The chaetal progression of only one species Hyalinoecia araucana has been studied within Hyalinoeciinae (Carrasco 1983). The major character of Hyalinoecia chaetal progression is the absence of provisional anterior pseudocompound falcigers in early developmental stages. Chaetae resembling transitional compound falcigers appear first. Anterior compound falcigers, an autapomorphy of onuphids (Paxton 1986), appear on the first chaetiger only at the 13-chaetiger stage. This mode is similar to the chaetal progression in eunicids. In Eunice valens, transitional compound falcigers form first at the 1-chaetiger stage and together with falcate simple chaetae are the only chaetae present up to the 4-chaetiger stage (Åkesson 1967).

Two modes reported within Onuphinae have been summarized by Paxton (1996, 2005). The members of the Onuphis group sensu Paxton (1986) (e.g. Hirsutonuphis, Kinbergonuphis, Mooreonuphis, Onuphis) demonstrate a very similar pattern of chaetal progression that we consider to be derived. Provisional anterior falcigers are the first chaetae that appear during development. They are replaced by permanent falcigers when the full number of anterior parapodia characteristic for adults of each species has developed. Provisional compound subacicular hooks are the precursors of the permanent simple subacicular hooks. These appear a few segments earlier than the first appearance of the permanent SAH and are replaced gradually by the latter. In fact, the provisional compound subacicular hooks are retained in the posteriormost parapodia even in some adults (Paxton 1996). Transitional compound falcigers are present between anterior modified parapodia and parapodia with subacicular hooks. They are present only in larvae and juveniles and completely disappear in adults.

Orensanz (1990) and Paxton (1996) stated that transitional compound falcigers (anterior provisional subacicular hooks sensu Paxton 1996) are the precursors and homologues of subacicular hooks of adults. We consider them to be the precursors of adult eunicid compound falcigers and have no homologues among the onuphid permanent chaetae, appearing only in larvae and juveniles. This is supported by the fact that these falcigers resemble the compound falcigers of adult eunicids and always appear before provisional compound subacicular hooks and permanent simple bidentate hooks in larvae and juveniles of onuphids and eunicids.

A third mode of development in onuphids was described for members of the Diopatra group (Paxton 2005). Species of Diopatra Audouin & Milne-Edwards, 1833 (Krishnan 1936; Allen 1959; Fadlaoui et al. 1995) are characterized by the presence of two types of anterior provisional chaetae: curved chaetae and wing-tipped capillary chaetae. These chaetae appear first in larval development in anterior parapodia and are later replaced by anterior permanent pseudocompound falcigers. Provisional simple subacicular hooks appear a few segments before the start of the permanent subacicular hooks (Allen 1959). Although provisional anterior curved and wing-tipped capillary chaetae have not been reported for Brevibrachium (Estcourt, 1966) (Paxton 1986) and Fauchaldonuphis Paxton, 2005 (Paxton 2005), the presence of simple provisional subacicular hooks links these species to Diopatra and supports the monophyly of the Diopatra group (Paxton 2005).

Development of branchiae

The first appearance of branchiae in M. stigmatis was not observed. Juveniles that leave the parental tube are abranchiate and only develop branchiae later. A comparison of branchial development in other onuphid species demonstrates a similar pattern. In all cases branchiae first appear in the median segments (e.g. chaetigers 5–10 in a 20-chaetiger specimen) subsequently, depending on the adult branchial distribution, disperse anteriorly and posteriorly as in Onuphis elegans (Blake 1975), Kinbergonuphis simoni (Hsieh & Simon 1987) and Diopatra marocensis (Fadlaoui et al. 1995) or disappear late in larval development and appear secondary in juveniles as in Mooreonuphis jonesi (Fauchald 1982b).

Fauchald (1982a) stated that the beginning of branchiae from chaetigers 5–10 was a plesiomorphic condition while origin of branchiae on or before chaetiger 4 or after chaetiger 10 as apomorphic conditions. Paxton (1986) rejected this hypothesis, and, based on the late origin of branchiae in Hyalinoecia, considered late origin of branchiae (after chaetiger 10) to be a plesiomorphic condition and the early origin (from chaetigers 1–9) to be apomorphic. Comparison of branchial origin during development in onuphids (Table V) supports the hypothesis of Fauchald (1982a), but apparently the transformation from plesiomorphic to apomorphic conditions must have occurred several times independently since most of the onuphid genera include species both with primitive and derived branchial distribution, so the identification of apomorphic and plesiomorphic conditions for the family as a whole becomes a disputable question.

Table V. A comparison of the development of branchiae among five species of onuphids.

Species	Stage when first branchiae appear	No. chaetiger with first branchiae in larvae	Range of chaetigers with branchiae in adults	Reference
Diopatra marocensis	18–20-chaetiger	5	4(5)–19(43)	Fadlaoui et al. 1995
Kinbergonuphis simoni	36-chaetiger	9–10	6(9)–20	Hsieh & Simon 1987
Mooreonuphis jonesi	10-chaetiger	6–8	29–85	Fauchald 1982b
Mooreonuphis stigmatis	Later than 24-chaetiger	?	16(20)– posterior	Present study
Onuphis elegans	9-chaetiger	6–7	1– posterior	Blake 1975

Editorial responsibility: Kenneth Halanych

Acknowledgements

We are grateful to Svetlana Maslakova and George von Dassow and the staff of the Friday Harbor Laboratories, University of Washington, for help with collecting and preserving specimens for the SEM study. We would like to thank Elena Vortcepneva from Moscow State University for providing the fixation protocol. Also, we are grateful to Scott Whittaker from the National Museum of Natural History, Smithsonian Institution, for assistance with SEM. This work was supported by the Smithsonian Institution fellowship program, the Russian Foundation for Basic Research, grant 06-04-48764 and the tuition and course-related expenses award, Comparative Embryology Course, Friday Harbor Laboratories, University of Washington.

Notes

Published in collaboration with the University of Bergen and the Institute of Marine Research, Norway, and the Marine Biological Laboratory, University of Copenhagen, Denmark