Long forgotten: Eunice woodwardi Baird, 1869 (Annelida, Eunicidae) revisited, with an insight on internal anatomy

Introduction

Eunice Cuvier, 1817 is the most species-rich genus of the annelid family Eunicidae Berthold, 1827 and comprises 250 valid species (Read & Fauchald, 2022). It is widely distributed and can be found in soft and hard substrates from the intertidal zone to the deep sea in all oceans (Hutchings, 1986). The diagnosis of the genus was traditionally based on a combination of several morphological characters, such as the presence of three prostomial antennae, a pair of palps, a pair of peristomial cirri, and a set of chaetal types that includes: limbate, pectinate and compound chaetae, and subacicular hooks (Orrhage, 1995). However, all these are plesiomorphic characters shared with other eunicid genera such as Euniphysa Wesenberg-Lund, 1949 (Lu & Fauchald, 2000) and even with members of other families such as Onuphidae Kinberg, 1865 (Zanol, Fauchald & Paiva, 2007). Furthermore, original descriptions of many Eunice species are often based on incomplete or a low number of specimens and therefore intraspecific variability is not well delimited. Consequently, many species are not characterized properly and this represents the main issue in assessing the true diversity of the genus (Miura, 1986; Fauchald, 1992). In this context, species of Eunice had traditionally been “informally” grouped according to characters lacking real systematic value, such as the branchial distribution patterns and the colour and teeth number of subacicular hooks (e.g., Ehlers, 1868; Hartman, 1944; Fauchald, 1970; Carrera-Parra & Salazar-Vallejo, 1998). In fact, recent phylogenetic studies on Eunicidae conclude that Eunice sensu lato is actually non-monophyletic (Zanol et al., 2010, 2021; Zanol, Halanych & Fauchald, 2014). Besides, the genus Leodice has recently been resurrected and several species of Eunice transferred there (Zanol, Halanych & Fauchald, 2014). Zanol et al. (2021) also suggest that some Eunice species correspond to a clade defined by branchiae starting late, ventral cirri with inflated base shaped as thick ventral ridges, left MxIV with dorsal teeth only, and anteriormost chaetigers bearing bidentate falciger chaetae with inconspicuous blade teeth. However, there is no consensus in placement of some species and whether there are reliable morphological characters to clearly delineate the involved genera (Zanol, Halanych & Fauchald, 2014; Molina-Acevedo & Carrera-Parra, 2017). Therefore, it seems that further phylogenetic analyses based in a wide sampling of species and molecular data are needed to clarify the actual systematics of species included in Eunice and Leodice (Zanol et al., 2021).

On the other hand, the general internal anatomy (e.g., nervous system, muscular system, circulatory system and nephridia) of a number of annelid families is well known (see Bartolomaeus & Purschke, 2005; Zanol, 2010), but only Ehlers (1868) studied in detail the digestive tract of the genus Eunice as well as other Eunicidae. Nevertheless, many anatomical features of the digestive tract of this genus still remain undescribed.

Eight species of Eunice sensu lato have been reported in the Iberian Peninsula: E. norvegica (Linnaeus, 1767), E. pennata (Müller, 1776), E. vittata (Delle Chiaje, 1829), E. purpurea Grube, 1866, E. roussaei Quatrefages, 1866, E. woodwardiBaird, 1869, E. schizobranchia Claparède, 1870 and E. dubitata Fauchald, 1974. Eunice woodwardi was collected by MacAndrew & Woodward (1864) during benthic sampling in the Ría de A Coruña (NW Spain). This species is morphologically very close to E. vittata mostly regarding the presence of tridentate subacicular hooks, that is a highly relevant diagnostic character. However, E. woodwardi has been largely ignored and overlooked after original description; for instance, later monographies on European polychaetes (e.g., Fauvel, 1923) do not even mention this species. Hartman (1959) questioned whether E. woodwardi was a synonym of E. vittata, and finally regarded it as indeterminable. Later, Fauchald (1992) re-examined the holotype of E. woodwardi and consider it as a valid species, also providing a redescription. Nevertheless, several key characters in Eunice, such as the maxillary formula were not described since this would imply dissection and consequently a potential deterioration of the only specimen available. Anyway, it seems that E. woodwardi fell into oblivion despite Fauchald’s redescription. This might also be due to E. vittata being considered as a cosmopolitan species and, therefore, it is likely that specimens of Eunice with tridentate subacicular hooks might have often been identified as E. vittata across the world. In fact, the presence of E. woodwardi in NW Spain also remained overlooked even in recent publications (e.g., Parapar et al., 1993; Parapar, Urgorri & Besteiro, 1996; Parapar & Moreira, 2009; Besteiro et al., 2018). In this context, Zanol et al. (2021) challenge the wide range of distribution traditionally attributed to several eunicids, suggesting that E. vittata might have a more restricted distribution.

In this study, we present an integrative approach to the taxonomy and anatomy of E. woodwardi in order to support the validity of the taxon. Additional anatomical information is provided after examination of the holotype and non-type material collected near the type locality; the original description and redescription were complemented with other characters not observed in the holotype, also assessing its intraspecific variability. The main features of the internal anatomy of E. woodwardi are described for the first time and the presence of epibiont ciliophorans on branchiae is also reported.

Materials and Methods

This work is based on the study of the holotype of E. woodwardi from off A Coruña (Galicia, NW Spain), along with 34 newly collected specimens collected in the nearby Ría de Ferrol (Table 1). Selected specimens have been deposited in the Museo Nacional de Ciencias Naturales, Madrid, Spain (MNCN) and the British Museum (Natural History), London, England (NHML). For comparative purposes, specimens identified as E. vittata from several Mediterranean locations were also examined: Two specimens from Naples, Italy (type locality; Museum of Natural History of Wroclaw, MNHW), five from Venice, Italy (Zoological Museum Hamburg, ZMH), two from Banyuls-sur-Mer, France (ZMH), five from Valencia, Spain (MNCN) and four from Mallorca, Spain. Type material could not be examined because there are no longer in existence (Fauchald, 1992, p. 337). The description of E. woodwardi holotype is based on Fauchald (1992) and is complemented with our observations. Additional observations of material collected in the Ría de Ferrol and E. vittata from Naples are also provided.

Results

Eunice woodwardiBaird, 1869

Eunice woodwardiBaird, 1869: 347; Fauchald, 1992: 343–345, Fig. 117, Tables 41–42.

Eunice vittata (Delle Chiaje). Parapar et al., 1993: 117–125 (non Delle Chiaje, 1829).

Material examined

Type material: Eunice woodwardiBaird, 1869. Holotype (BM(NH) ZH 1863.8.19.13; Ría de A Coruña; Table 1).

Non-type material: 34 specimens (MNCN 16.01/19142 to 16.01/19169; Ría de Ferrol; Table 1).

External morphology

Holotype

Incomplete specimen, 49 mm long, 5 mm wide, with 59 chaetigers. Mature female, oocytes first observed in chaetiger 18. Body slightly flattened dorsoventrally, whitish (Figs. 1A, 1B); original colouration not preserved.

Prostomium nearly as wide as peristomium but shorter (Fig. 1D). Prostomial lobes frontally rounded and obliquely divided (Figs. 1B, 1D). Prostomial appendages equally separated from each other and arranged in horseshoe shape; all antennae with ring-shaped ceratophores; ceratostyles slender, digitiform, smooth, lacking distinct articulations or constrictions (Figs. 1A–1D). Median antenna about 1.2 times as long as lateral antennae; palps one third as long as lateral antennae (Figs. 1A–1D). Eyes not seen. Peristomium cylindrical; anterior ring longer than posterior one (Fig. 1C); peristomial cirri digitiform, smooth, slightly thinner than palps (Fig. 1C). Maxillary apparatus and mandibles not observed; mandible distal end just protruding from oral cavity (Fig. 1D).

Branchiae pectinate, branchial stem longer than dorsal cirri (Fig. 1C), present from chaetiger 3 to 39; first branchia with two filaments (Fig. 1C); up to 12 filaments per branchia in following chaetigers. Chaetigers 15–24 showing maximum number of filaments; last chaetigers of branchial region with 1–2 filaments. Branchial filaments of mid-branchial region longer or about as long as dorsal cirri.

Parapodia sub-biramous. Dorsal cirri smooth, digitiform, tapering. Ventral cirri short with a digitiform tip, inflated basally from chaetiger 3 to 40. Dorsal and ventral cirri decreasing in length from anterior to posterior parapodia (Table S1).

Pre- and postchaetal lobes low, transverse folds. Prechaetal lobes folds covering bases of compound falcigers. Acicular lobes truncate. 3–4 notoaciculae, thin, distally bent; two (sometimes three) yellow neuroaciculae, one larger than the other, tapering with blunt tips, curved distally and protruding from acicular lobe, never T-shaped. Chaetae including 5–13 limbate, 1–3 pectinate, 5–20 compound falcigers and 1–5 tridentate subacicular hooks. Limbate and pectinate chaetae arranged in a bundle dorsal to neuroaciculae. Limbate chaetae elongated, marginally serrated, distally curved and tapering. All pectinate chaetae heterodont, about 0.3–0.4 times as long as limbate chaetae; one external tooth three times as long as others. Compound falcigers ventral to neuroaciculae; shafts distally inflated and marginally serrated. Blades bidentate; proximal tooth slightly larger than distal tooth, triangular, perpendicular to blade axis; distal tooth curved dorsally; distal end protected by elongated guard, marginally serrated and provided with conspicuous apical mucro. Number of limbate and compound falcigers decreasing from anterior to posterior chaetigers (Tables S2, S3). Subacicular hooks from chaetiger 31, yellowish, tridentate with teeth in a crest, distal end protected by rounded guards. Number of hooks increasing from 1 to 5 from chaetiger 31 to 59. Hooks almost hidden within parapodia in anterior chaetigers and protruding conspicuously in last chaetigers.

Non-type material

Largest complete specimen 75 mm long, 4 mm wide, with 112 chaetigers; mean length 30 mm (standard deviation, S.D. = 18), mean width 1.2 mm (S.D. = 0.9 mm), with 37–112 chaetigers.

Body slightly flattened dorsoventrally; most specimens whitish, original colouration not preserved in alcohol. Original colouration was observed in three specimens (Figs. 2–3, 4A, 4B). One specimen studied alive showing colouration consisting of two broad dorsal dark red bands per segment separated by bands of much lighter tone, consecutive segments separated by a dark red thin band (Figs. 2–3, 4B); bands from peristomium to posterior body end, slightly fading in tone from posterior branchial region to posterior body half depending on specimen and preservation state. One preserved specimen showing a different colouration pattern, consisting of transverse dark bands much more faded, and bands of lighter tone wider (Fig. 4A).

Prostomium nearly as wide as peristomium but shorter (Figs. 2C, 2E, 4C, 5A). Prostomial lobes frontally rounded and obliquely divided (Figs. 2C, 2E, 4D). Prostomial appendages equally separated from each other, arranged in horseshoe shape; all antennae with ring-shaped ceratophores (Figs. 5B, 5D); ceratostyles slender, digitiform, smooth and lacking distinct articulations or constrictions (Figs. 2, 4C, 4D, 5A, 5C, 5E, 5F). Median antenna about 1.2 times as long as lateral antennae; palps one third as long as lateral antennae (Figs. 2A, 4C, 4D, 5E). A pair of dark eyes between lateral antennae and palps (Figs. 2A, 2B, 2D, 3A, 4A). Peristomium cylindrical; anterior ring longer than posterior one (Figs. 2B, 2D, 4C); peristomial cirri digitiform, smooth, thinner than palps (Figs. 2B, 2D, 3A, 4C). Mandibles flat (Fig. 6A). Maxillary formula: MxI: 1+1, MxII: 8+9–10, MxIII: 9–11+0, MxIV: 10–11+11, MxV: 1+1, MxVI: absent. Mx III behind left MII. Left MxIV wider than long. MxV fang-shaped (Fig. 6B).

Branchiae pectinate, stem longer than dorsal cirri (Figs. 2A, 3C, 4C, 6C), from chaetiger 3 to 36–46 (<55% of body chaetigers); first branchia with 1–2 filaments (Figs. 4C, 5E, 7A); up to 9–14 filaments per branchia in following chaetigers. Chaetigers 15–25 with maximum number of filaments (Figs. 2A, 3A–3C, 6C, 7B–7D), last branchiate chaetigers with 1–2 filaments (Figs. 3A, 3D). Branchial filaments longer or about as long as dorsal cirri in mid-branchial region; filaments showing two longitudinal fringes of discocilia (Figs. 6D, 6E).

Parapodia sub-biramous. Dorsal cirri smooth, digitiform, tapering. Ventral cirri short with a digitiform tip (Figs. 7A–7E), inflated basally from about chaetiger 3 to 40; starting in chaetiger 3 in all specimens (Fig. 7C). Variation in length and width of dorsal and ventral cirri depending on specimen size, decreasing in length from anterior to posterior parapodia (Table S1). Lateral interramal red dots present in almost all body segments.

Pre- and postchaetal lobes low, transverse folds. Prechaetal lobe fold covering proximal third of compound falcigers shaft. Acicular lobes truncate (Figs. 7A–7C). 3–4 notoaciculae, thin, distally bent (Figs. 7A–7C, 7E); two (sometimes three) yellow neuroaciculae, one larger than the other, all neuroaciculae tapering with blunt tips, curved distally and protruding from acicular lobe, never T-shaped (Figs. 7A–7E, 7G, 8F, 8G). Chaetae including 3–14 limbate, 1–4 pectinate, 3–23 compound falcigers and 1–5 tridentate subacicular hooks. Limbate and pectinate chaetae arranged in a bundle dorsal to neuroaciculae (Figs. 7A, 7B). Limbate chaetae elongated, marginally serrated (Figs. 8A–8D), distally curved and tapering (Figs. 7A, 7B). All pectinate chaetae heterodont (Fig. 8E), about 0.3–0.4 times as long as limbate chaetae; 7–9 teeth and one external tooth three times as long as others. Compound falcigers ventral to neuroaciculae (Figs. 7A, 7B); shafts distally inflated and marginally serrated (Figs. 7F, 9A, 9B). Blades bidentate (Figs. 7F, 9A–9C); proximal tooth slightly larger than distal tooth, triangular, perpendicular to blade axis; distal tooth curved dorsally (Fig. 9C); blade distal two thirds protected by elongated guard, marginally serrated (Figs. 9A–9C) and provided with conspicuous apical mucro (Figs. 7F, 9D). Number of limbate and compound falcigers decreasing from anterior to posterior chaetigers (Tables S2, S3). Subacicular hooks from chaetigers 16–31 to posterior region (Fig. 7H), ventral to neuroaciculae (Figs. 7C–7E), yellowish, tridentate with teeth in a crest, distal end protected by rounded guards (Figs. 8H–8J); size decreasing towards posterior region. Number of hooks size-depending, numbering 3 at least; increasing from 1 up to 5 from chaetiger 25–30 to middle-posterior region and then decreasing to 1–2 towards posterior region. Hooks almost hidden within parapodia in anterior chaetigers and protruding conspicuously in last chaetigers.

Pygidium with two pairs of cirri, one pair two times as long as other, smooth, non-articulated.

Cuticular glandular pores present throughout body surface (Fig. 9E).

Internal anatomy

The digestive tract is clearly regionalized and consists of: (1) pharynx (provided with mandibles and maxillae), (2) oesophagus, (3) stomach, (4) fore intestine, (5) mid-intestine, and (6) hind intestine (Figs. 10, 11A, 11B). The pharynx (Figs. 10A, 11A, 11B, 12A) is located ventrally and provided with a pair of rounded structures (g, Figs. 11A, 11B; see Discussion). The oesophagus is elongated and straight or slightly folded in shape depending on the state of pharynx protraction (Figs. 10A, 10B, 11A–11C, 12A, 12B, 13A, 13B, 14A). The oesophagus is connected to a highly muscular stomach (Figs. 10A, 10B, 10D, 11A–11C, 12C, 13C, 13D, 14A, 14B). It is followed by the fore intestine and the mid-intestine, ending in the hind intestine (Figs. 10A–10C, 11A–11C, 12D–12F, 13E, 13F, 14C, 15); the latter is a long and narrow tube provided with a very thin wall (Figs. 13F, 15).

The body musculature is well developed, particularly in the pharynx and body wall (Figs. 12, 15). It is composed by circular, dorsal and ventral longitudinal and ventral oblique muscles (Fig. 15). The longitudinal musculature is divided into four bands, two arranged dorsally and two ventrally, the latter flanking the ventral nerve cord at both sides (Figs. 13A, 13C, 14C, 14D, 15).

The circulatory system includes two longitudinal ventral and one (sometimes two) dorsal vessels (Figs. 13F, 14D, 14E). Blood masses (blood sinuses) attached to the digestive tract were also observed, being especially abundant in the oesophagus, stomach, and intestine (Figs. 10C, 10D, 11C).

A large glandular mass was observed basally in each parapodium closely associated to the chaetal bundle (Figs. 10C, 10D, 11D, 15).

The central nervous system comprises a well-developed brain (Figs. 16A–16E), supra-oesophageal nerves (Figs. 16A, 16C, 16E) and the typical ventral nerve cord, with pair of ganglia in same segment almost fused to each other. Several sensory organs were observed: (1) a pair of eyes located slightly behind lateral antennae (Figs. 16A, 16C, 16E), (2) a pair of nuchal organs in posterior half of anterior peristomium ring (Figs. 16C–16G), and (3) ciliary areas located in proximal third of dorsal cirri in all chaetigers (Figs. 17A–17D); the aforementioned cilia correspond to discocilia (Figs. 17C, 17D).

Another ciliary area of unknown function was observed ventrally to ventral parapodial cirrus (Figs. 17A, 17E). The epidermis nearby this area in mid-body segments has several pores arranged in two groups next to each other; the larger pores are about 1 µm in diameter and the smaller ones 0.2–0.3 µm. Each pore is provided with a cilium (Figs. 17E, 17F).

Nephridia appear as glandular masses arranged metamerically that were observed in most of body segments apart from the anteriormost and posteriormost segments (Fig. 18). These are located ventro-laterally between the ventral longitudinal musculature and the glandular masses associated with chaetal bundles, measuring 70–80 µm (Figs. 18A, 18B) and directly connected with nephridiopores, each 3–4 µm in diameter (Fig. 18C).

The presence of gametes in coelomic body cavity was observed with LCM, micro-CT and HIS (Figs. 1A, 10C, 10D, 17G); specimens observed with micro-CT and HIS were collected in September-November. The specimen subjected to HIS bears gametes at an early stage of maturation and corresponds probably to a male due to the small gamete size (about 4–5 µm in diameter), high numbers and grouping shape (Fig. 17G). In anterior body half, gametes seem to be associated to the parapodial area, whereas in posterior half, gametes fill all the coelomic cavity. Muscular bands are less developed in posterior body half, where gametes are especially abundant; this region also lacks glandular masses associated with chaetae, blood vessels and nephridia (Fig. 17G). Specimens observed with LCM and micro-CT (Figs. 1A, 10C, 10D) are females with oocytes in an advanced stage of maturation due to their large size (150–200 µm). It was not possible to identify the location of gonads in the studied specimens; >50% of specimens were incomplete, with the posterior end missing.

Epibiosis

Ciliophorans were observed attached to the surface of branchial filaments (Fig. 19) but not showing any defined pattern of attachment or distribution.

Distribution and ecology

The type locality corresponds to Ría de A Coruña (Galicia, NW Spain). Unfortunately, Baird (1869) did not provide neither the coordinates nor depth or abiotic characteristics where this specimen was collected. Non-type specimens collected in the Ría de Ferrol were found from the intertidal to 26.5 m depth, in a wide range of bottom types, from coarse (gravel) to fine (sandy mud and mud) sediments (Table 1).

Eunice vittata (Delle Chiaje, 1829)

Nereis vittataDelle Chiaje, 1829: 195.

Eunice vittata (Delle Chiaje). Grube, 1850: 293; Fauchald, 1992: 337–339, Fig. 115a-i, Tables 18, 41–42.

Material examined

Non-type material (Table 1): 2 specimens (MNHW; Naples, Italy), 5 specimens (ZMH-V 12932; Venice, Italy), 2 specimens (ZMH-P 14276, Banyuls-sur-Mer, France), 5 specimens (MNCN 16.01/2677 and 16.01/2723; Valencia, Spain) and 4 specimens (SPR04-03, PAD04-03, CBR01-06 and CBR01-11, Mallorca, Spain).

External morphology (specimens from Naples)

Two specimens; one complete: 24 mm long, 2 mm wide, with 84 chaetigers, and one incomplete: 23 mm long, 2 mm wide, with 80 chaetigers.

Body slightly flattened dorsoventrally; original colouration not preserved in alcohol (Figs. 20A, 20B).

Prostomium nearly as wide as peristomium but shorter. Prostomial lobes frontally rounded. Prostomial appendages equally separated from each other, arranged in horseshoe shape; all antennae with ring-shaped ceratophores; ceratostyles slender, digitiform, smooth, lacking articulations but with poorly marked constrictions (Figs. 20C, 20D). A pair of dark eyes between lateral antennae and palps. Peristomium cylindrical; peristomial cirri digitiform, smooth.

Branchiae pectinate, stem longer than dorsal cirri, present from chaetiger 3 to 27–29 (<55% of body chaetigers); first branchia with 3 filaments (Fig. 20C); up to 7 filaments per branchia in following chaetigers. Last branchiate chaetigers with one filament. Branchial filaments longer or about as long as dorsal cirri in mid-branchial region chaetigers.

Parapodia sub-biramous. Dorsal cirri smooth, digitiform, tapering. Ventral cirri short with a digitiform tip, inflated basally from about chaetiger 3 to 27–29, in all specimens starting in chaetiger 3. Dorsal and ventral cirri decreasing in length from anterior to posterior parapodia (Figs. 20C, 20D). Lateral interramal red dots present in almost all body segments.

Two yellow neuroaciculae, one larger than the other; all neuroaciculae tapering with blunt tips, curved distally and protruding from acicular lobe, never T-shaped. Chaetae including 2–11 limbate, 1–3 pectinate, 3–23 compound falcigers and 1–5 tridentate subacicular hooks. Limbate and pectinate chaetae arranged in a bundle dorsal to neuroaciculae. Limbate chaetae elongated, marginally serrated, distally curved and tapering. All pectinate chaetae heterodont, about 0.3–0.4 times as long as limbate chaetae; number of teeth not observed, one external tooth three times as long as others. Compound falcigers ventral to neuroaciculae; shafts distally inflated and marginally serrated. Blades bidentate; proximal tooth slightly larger than distal tooth, triangular, perpendicular to blade axis; distal tooth curved dorsally; blade distal two thirds protected by elongated guard, marginally serrated; mucro absent. Number of limbate and compound falcigers decreasing from anterior to posterior chaetigers (Tables S2, S3). Subacicular hooks from chaetigers 21–22 to posterior region, ventral to neuroaciculae, yellowish, tridentate with teeth in a crest, distal end protected by rounded guards; size decreasing towards posterior region. Maximum number of hooks in posteriormost chaetigers. Hooks almost hidden within parapodia in anterior chaetigers and protruding conspicuously in last chaetigers.

Discussion

Conclusions

Our examination of the holotype and additional material near the type locality confirms that E. woodwardi is a valid species and different to E. vittata. Eunice woodwardi is distinguished by the non-articulated and non-constricted cephalic appendages, the maxillary formula, the range of branchial distribution, the maximum number of branchial filaments, the presence of an apical mucro in the guard of falciger chaetae blades, and the number of teeth in pectinate chaetae. Eunice woodwardi is at least present in the rias of A Coruña and Ferrol, and we suggest that previous reports of E. vittata from the NW Iberian Peninsula should be reviewed. The integrative use of different anatomical techniques for the study of the internal anatomy has confirmed previous observations in the genus Eunice sensu lato and allowed reporting some previously unknown structures such as the ciliated areas located below ventral cirrus. The presence of ciliophoran epibionts on branchiae is also reported.

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