COI analysis and haplotype network.

The alignment of COI sequences obtained in the present study and those from GenBank and BOLD comprised 267 bp with 106 (39.7%) variable sites, of which 93 (34.8%) sites were parsimony informative. The Bayesian analysis resulted in a partially resolved consensus tree (Fig 1B).

The analysis revealed two sister groups among specimens from northern Europe identified by morphology as S. bombyx: one from the North and the Norwegian seas (referred to as S. cf. bombyx), and another from Brittany, northern France, and the Bay of Biscay, northern Spain (referred to as S. cf. convexus). The p-distances among individuals of these groups ranged from 12.73% to 14.98%. The ingroup p-distance values ranged from 0.00% to 1.5% and from 0.75% to 1.87%, respectively.

Horned Spiophanes from the North Pacific formed a well supported clade (PP = 1) closely related to the clade of horned Spiophanes (S. cf. bombyx—S. cf. convexus) from northern Europe. The relationships within the North Pacific clade were partially resolved, with one clade well supported (PP = 0.97), comprising one group of specimens from California, USA, and Baja California Norte, Mexico (referred to as S. norrisi according to the type locality of the species in Baja California Sur, Mexico), and another group S. uschakowi from the Sea of Japan (East Sea). The p-distances between individuals of these groups ranged from 6.74% to 8.99%. The rest of the specimens within the North Pacific clade did not form a monophyletic group, but, according to the results of the five-genes analysis (see below), they are referred to S. hakaiensis n. sp. The three groups of North Pacific Spiophanes did not share any haplotype (Fig 1A).

Two sister groups were revealed among specimens from northern Europe identified by morphology as S. kroyeri: one from the Barents Sea and northern part of the North Sea (tentatively referred to as S. cf. kroyeri), and another from the northern and central parts of the North Sea and from Bay of Biscay, northern Spain (tentatively referred to as S. cf. cirrata). The p-distances between individuals of these groups ranged from 6.37% to 8.61%. The ingroup p-distance values ranged from 0.00% to 1.12% and from 0.75% to 1.5%, respectively.

Five-genes analysis (COI, 16S, 18S, 28S, and Histone 3).

The five-genes analysis did not include sequences provided by Meißner & Blank [111] because they were too short and did not fit the accepted criteria (COI > 500 bp, 18S > 1600 bp). The combined alignment, with gaps excluded, comprised in total 3018 bp, including 244 bp (93.5%) for 16S rDNA, 1656 bp (99%) for 18S rDNA, and 287 bp (98.6%) for 28S rDNA, 297 bp (100%) for Histone 3 and 534 bp (100%) for COI. It contained 677 (22.4%) variable sites, of which 615 (20.4%) sites were informative. The average p-distances for the individual gene fragments between taxa are given in S3–S7 Tables. The Bayesian analysis resulted in a fully resolved tree with high support of all branches (Fig 2B).

The analysis revealed two sister groups among specimens from northern Europe identified by morphology as S. bombyx. Specimens from the North and the Norwegian seas (referred to as S. cf. bombyx) were genetically close to single examined specimen of S. bombyx from Venice Lagoon, Adriatic Sea, Italy (p-distances ranging from 00% for 28S to 2.46% for 16S). Specimens from Brittany, northern France, and the Bay of Biscay, northern Spain, are referred to as S. cf. convexus. The p-distances between individuals of S. cf. bombyx and S. cf. convexus ranged from minimal 0.3% for 18S rDNA to maximal 14.07% for COI. The maximum ingroup p-distance values were for COI sequences: 0.94% and 1.69%, respectively.

Horned Spiophanes from the North Pacific formed three distinct groups within a clade sister to the European horned Spiophanes. The groups comprised S. uschakowi from the Sea of Japan (East Sea), worms from Alaska to San Francisco Bay, California, described below as S. hakaiensis n. sp., and worms from San Francisco Bay, California, and Baja California Norte, Mexico, referred to S. norrisi. Spiophanes uschakowi and S. hakaiensis n. sp. formed a clade sister to S. norrisi. The p-distances between individuals of S. uschakowi and S. hakaiensis n. sp. ranged from minimal 0.91% for 18S rDNA to maximal 7.68% for COI. The maximum ingroup p-distance values were for COI sequences: 0.94% and 1.12%, respectively.

Three groups were distinguished among specimens identified by morphology as S. kroyeri: one from the Barents Sea and northern part of the North Sea (referred to as S. cf. kroyeri), second from the northern and central parts of the North Sea and from the Bay of Biscay, northern Spain (referred to as S. cf. cirrata), and a third from the Southern Ocean (referred to as S. aff. kroyeri). The S. cf. kroyeri group appeared sister to S. cf. cirrata. The average p-distances for the individual gene fragments between these groups are given in S3–S7 Tables. Spiophanes aff. kroyeri appeared distant from the clade S. kroyeri—S. cf. cirrata.

Two sister groups were revealed among specimens identified by morphology as S. duplex: one from California, USA (referred to as S. duplex), and another from Paraná, Brazil (referred to as S. soederstroemi). The p-distances between individuals of these groups ranged from 0.18% for 18S to 5.05% for Histone 3.

Horned Spiophanes with metameric nuchal organs formed a clade sister to the clade of Spiophanes with bell-shaped prostomium and nuchal organs as a pair of long parallel ciliary bands extending over about 15 anterior chaetigers (Fig 2B).

The analysis showed identity (p-distance = 0.00%) of 18S and 28S rDNA sequences of Trochochaeta multisetosa from Norway, Sweden and California, USA.

An additional analysis showed identity (p-distance = 0.00%) of our sequence of S. berkeleyorum from Southern California and short fragments of 18S rDNA (490 bp) of S. berkeleyorum from Northern California obtained by Meissner & Blank [111] (Fig 2A).

Divergence time estimation.

The alignment of COI sequences obtained in the present study and those from GenBank and BOLD comprised 534 bp with 170 (31.8%) variable sites, of which 160 (94.1%) sites were parsimony informative. The BEAST analysis resulted in a fully resolved consensus tree (Fig 3) with the same topology as the tree from the five-genes Bayesian analysis (Fig 2B). The analysis suggested that the origin of the most recent common ancestor of horned Spiophanes with metameric nuchal organs was around 11.1 mya (95% HPD: 5.1–19.0 mya) and that the divergence of the North Atlantic and North Pacific lineages was around 7.9 mya (95% HPD: 4.1–13.3 mya). The North Atlantic lineage was estimated to have diverged 4.8 mya (95% HPD: 2.2–8.6 mya), resulting in the origin of S. cf. bombyx and S. cf. convexus. The North Pacific lineage was estimated to have diverged first by the isolation and speciation of S. norrisi 1.7 mya (95% HPD: 2.3–1.0 mya), then by the isolation and speciation of S. uschakowi and S. hakaiensis n. sp. 1.3 mya (95% HPD: 2.0–0.7 mya).

Spiophanes bombyx (Claparède, 1870).

Spio bombyx Claparède, 1870a [9]: 485–487, pl XII, Figs 2, 2A–2H; [10]: 121–123, pl XII, Figs 2, 2A–2H. Fide Mesnil [11].

Spiophanes bombyx: Fauvel [140] (Part.): 41. Rullier [141]: 209. Bhaud [142]: 235; [143]: 290–291, 306 (larval phenology). Katzmann [144]: 133. Cognetti-Varriale & Zunarelli-Vandini [145]: 42. Gambi & Giangrande [146]: 851. Sardá [147]: 346. Somaschini [148]: 223. Martin et al. [149]: 5. Simboura & Nicolaidou [150]: 87. Aleffi et al. [151]: 221. Ayari & Afli [152]: 89; [153]: 73. Dagli et al. [154]: 155. Castelli et al. 20[155]08: 357. Meißner [8] (Part.): 54–58. Meißner & Blank [111] (Part.): 7–11, Figs 3 and 4C. Mutlu et al. [156]: 32. Lorenti et al. [157]: 331. Serrano et al. [158]: 339. Çinar et al. [159]: 2114; [160]: 1462. Mikac [161]: 123. Delgado-Blas et al. [162] (Part.): 340.

Type material. ITALY, Tyrrhenian Sea, Gulf of Naples, presumably 40.8287°N, 14.2474°E, types lost.

Synopsis. Up to 38 mm long, 1 mm wide for 120 chaetigers. Prostomium with long fronto-lateral horns, posteriorly pressed into chaetiger 1 but not extending over it as caruncle. Occipital antenna absent. Nuchal organs metameric, on a series of anterior chaetigers; first pair of metamers as oblique ciliary bands from prostomium to end of chaetiger 2; succeeding metamers shorter. Sabre chaetae in neuropodia from chaetiger 15. Hooks in neuropodia from chaetiger 15, usually quadridentate, with small subterminal hood. Pygidium with small ventral fleshy pad and one pair of lateral cirri. Glandular organs in chaetigers 5–14, largest in chaetigers 7 and 8, smallest in chaetiger 9, gradually increasing in size from chaetiger 10 to chaetigers 12–13, slightly smaller again in chaetiger 14. Internal fibres long and coiled in glandular organs in chaetigers 5–8, straight, shorter and thinner in chaetigers 9–14. Organs on chaetigers 5, 7 and 8 each opening to exterior via semicircular to suboval slit around large fiber spreader, on chaetiger 6 via small round hole, on chaetigers 9–14 via large vertical slit. Frontal edge of each fiber spreader on chaetigers 5, 7 and 8 entire, rounded to blunt, or with variously developed middle depression vaguely separating two rounded lobes. Digestive tract with gizzard-like structure. Main dorsal blood vessel with heart body. Nephridia from chaetiger 15 onwards. Gonochoristic. Oocytes lentiform, each with honeycombed envelope and 18–20 cortical alveoli regularly arranged in a peripheral circle. Spermatids interconnected in tetrads. Spermatozoa short-headed aqua-sperm with plate-like acrosomes. Fertilization in sea water. Larval development holopelagic, planktotrophic. Larvae with two pairs of red eyes on prostomium and a midventral ciliated pit on chaetiger 2.

Remarks. In the original description of S. bombyx, Claparède ([9]: 485, as Spio bombyx) noted that “It does not seem rare in Naples. At least, it lives in numerous societies. I received hundreds of them one day, lodged side by side in tubes of black mud.” He reported worms up to 38 mm long, 1 mm wide. The type material of S. bombyx does not exist any longer, probably because Claparède did not deposit the examined specimens at any museum. After Claparède, the species has rarely been reported from Italy and seems not to be very common in the Gulf of Naples at present. We were unable to find a sequence of S. bombyx from the Mediterranean in the literature. We collected one individual of S. bombyx from Venice Lagoon, Italy, and sequenced it for 16S and 28S gene fragments (see Table 1).

Morphological examination and molecular analysis of horned Spiophanes performed in the present study discovered two sibling species in northern European waters which we refer to as S. cf. bombyx and S. cf. convexus. Their morphology, taxonomy and the distribution are discussed below. It seems plausible that S. bombyx does not occur in northern European waters and that its distribution is limited to the Mediterranean only. To avoid confusion in identification of specimens based on morphological characters only, especially in areas of possible overlap of the species, and until more molecular data has been obtained from specimens from the Gulf of Naples, we suggest that northern European horned Spiophanes are referred to as S. bombyx aggregate (S. bombyx agg.), rather than to a particular species.

Distribution. Mediterranean Sea (Fig 4A).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. Maps showing records of Spiophanes spp. in North Atlantic Ocean and Mediterranean Sea.

(A) Spiophanes bombyx (Claparède, 1870) (red and green symbols): red star–type locality: Gulf of Naples, Tyrrhenian Sea, Italy; red triangle–single specimen sequenced in the present study; green circles–adults identified based on the morphology only. Spiophanes cf. bombyx (yellow and pink symbols): pink triangles–specimens sequenced in the present study; pink squares–specimens sequenced by Meißner & Blank [111]; yellow circles–adults identified based on the morphology only. Spiophanes convexus (turquoise symbols): turquoise star–type locality of S. convexus: Meira, Pontevedra, Galicia, Spain; turquoise rhomb–S. cf. convexus sequenced by Aylagas et al. ([121], as S. bombyx); turquoise triangles–S. cf. convexus sequenced in the present study; turquoise circles–adults identified based on the morphology only. (B) Spiophanes kroyeri Grube, 1860 (red, green and yellow symbols): red star–type locality: Greenland Sea, off Iceland; yellow circles–adults identified based on the morphology only; green triangles–S. cf. kroyeri sequenced in the present study; green square–S. cf. kroyeri sequenced by Meißner & Blank ([111], as S. kroyeri). Spiophanes cirrata M. Sars in G.O. Sars, 1872 (turquoise symbols): turquoise star–type locality: Drøbak, Oslofjord, Norway; turquoise squares–S. cf. cirrata sequenced by Meißner & Blank ([111], as S. kroyeri); turquoise rhombs–S. cf. cirrata sequenced by Aylagas et al. ([121], as S. kroyeri). Spiophanes reyssi Laubier, 1964: pink star–type locality: NE Lacaze-Duthiers, Gulf of Lion, Mediterranean France.

https://doi.org/10.1371/journal.pone.0234238.g004

Spiophanes cf. bombyx (Claparède, 1870). Figs 5–8.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. Adult morphology of Spiophanes cf. bombyx (Norway, MIMB 36701).

(A), Chaetigers 4–20, dorsal view, showing light brownish lateral sides of chaetigers 9–14. (B) Chaetigers 5–16, ventral view, showing arrangement of glandular organs in chaetigers 5–14 and light brownish lateral sides of chaetigers 10–14. (C) Anterior end, ventral view. (D) Posterior end, left lateral view. Abbreviations: ch5–ch15 –chaetigers 5–15; fh–fronto-lateral horn; lp–lateral peristomial lip; py–pygidial cirrus; sp–notopodial spine with recurved distal end. Scale bars: A– 300 μm; B– 200 μm; C, D– 50 μm.

https://doi.org/10.1371/journal.pone.0234238.g005

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. Chaetal morphology of Spiophanes cf. bombyx (Norway, MIMB 36701).

(A) Crook-like spine in neuropodium of chaetiger 1. (B) Hooded hooks and a fragment of sabre chaeta from neuropodium of a middle chaetiger, lateral view. (C) Hooded hooks from neuropodium of a middle chaetiger, frontal view. (D) Hooded hooks, hair-like alimbate capillary chaeta, and a sabre chaeta from neuropodium of a posterior chaetiger, lateral view. (E) Simple capillary chaeta and two spines with recurved distal ends from posterior notopodia. Abbreviations: ca–capillary chaeta; hh–hooded hook; ho–subdistal hood below main fang of hook; sa–ventral inferior sabre chaeta. Scale bars: A, E– 20 μm; B–D– 10 μm.

https://doi.org/10.1371/journal.pone.0234238.g006

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. Glandular organs of Spiophanes cf. bombyx (Norway, MIMB 36701).

Right-side organs in left lateral view; formalin-fixed specimen stained with an alcohol solution of methyl green. (A) arrangement of glandular organs in chaetigers 5–14; organs withdrawn from chaetigers 10 and 12. (B) Chaetiger 5. (C) Chaetiger 7. (D) Chaetiger 9. (E) Chaetiger 13. (F) Chaetiger 14. Abbreviations: ch5–ch13 –chaetigers 5–13; fi–fibers produced by glandular organs. Scale bars: A– 100 μm; B–F– 50 μm.

https://doi.org/10.1371/journal.pone.0234238.g007

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 8. Gametes of Spiophanes cf. bombyx (Norway, MIMB 36701).

Formalin-fixed specimens stained with an alcohol solution of methyl green. (A) Oocytes in chaetigers 21–25; right-side coeloms in left lateral view. (B) Coelomic oocytes in chaetigers 20 and 21. (C–E) Coelomic oocytes of different shape. (F) Coelomic spermatozoa. Abbreviations: ca, cortical alveoli; en, oocyte envelope; n, nucleus; nu, nucleolus. Scale bars: A– 300 μm; B–E– 20 μm; F– 10 μm.

https://doi.org/10.1371/journal.pone.0234238.g008

Spiophanes bombyx: McIntosh [15]: 182–186; [16]: pl XCIII, Fig 1, pl XCVI, Fig 14, pl XCVII, Fig 6, pl XCVIII, Fig 10, pl CV, Fig 9, pl CVI, Fig 9, pl CVII, Fig 16. Söderström [21]: 243–244, Fig 135. Fauvel [140]: 41, Figs 14a–i. Thorson [25]: 91–92, Fig 44 (larval morphology). Hannerz [163]: 33–36, Fig 9 (larval morphology). Hartmann-Schröder [164]: 327–329, Fig 112; [165]: 341–342, Fig 156. Böggemann [32]: 121. Meißner [8] (Part.): 54–58, Fig 35. Meißner & Blank [111] (Part.): 7–11, Figs 2, 4A, B, D. Delgado-Blas et al. [162] (Part.): 340.

Adult morphology (based on specimens from the Norwegian and North Seas, and around the British Isles and Iceland). McIntosh [15] noted that worms from St Andrews, Scotland, UK, were about 3 inches (76 mm) long for about 180 chaetigers; Meißner & Blank [111] examined specimens 0.2–1.5 mm wide.

Worms examined in the present study 0.3–1.5 mm wide, up to 30 mm long for 120 chaetigers. Pigmentation absent on body and palps. In large formalin-fixed specimens, lateral sides of chaetigers 9–14 (until chaetigers 16–17 in large specimens) light brownish-red due to fixed internal content of large, probably glandular, epithelial cells (Fig 5A and 5B).

Prostomium triangular, wide anteriorly, with a pair of long, distally pointed fronto-lateral horns (Fig 5C), posteriorly narrowed, pressed into chaetiger 1 but not extending over as a caruncle. Peristomium with narrow lateral lips closely applied to lateral sides of prostomium (Fig 5C), and a small ventral lip. Two pairs of small dark red eyes usually present; occasionally eyes absent. Occipital antenna absent. Palps as long as 7–15 chaetigers.

Nuchal organs metameric, at least on 20 anterior chaetigers, fewer in small individuals. First pair of metamers long, oblique ciliary bands extending from posterior part of prostomium to end of chaetiger 2, shorter in small individuals; posterior ends of metamers set wider apart. Succeeding metamers shorter, each extending from nototroch over posterior half of chaetiger, oriented parallel to body axis until chaetigers 13–14, oblique on succeeding chaetigers.

Notochaetae capillaries, long on four anterior chaetigers and on posterior chaetigers, shorter on middle chaetigers. Neuropodia of chaetiger 1 each with 1–2 heavy recurved, crook-like spines in addition to capillaries (Fig 6A). Notopodia of 5–10 posterior chaetigers each with 1–3 (usually one) long recurved spines in addition to capillaries (Fig 6E).

Sabre chaetae in neuropodia from chaetiger 15, one or rarely two in a tuft, with narrow limbation and fine granulation on distal part (Fig 6B–6D).

Hooks in neuropodia from chaetiger 15, up to 10 in a series, accompanied by inferior sabre chaetae throughout body and 1–2 short hair-like alimbate capillaries in 5–10 posterior chaetigers; hair-like capillaries in posterior neuropodia usually situated in upper and/or lower parts of hook row (Fig 6D). Hooks usually quadridentate, with three small upper teeth above main fang and small subterminal hood below main fang (Fig 6B–6D); median upper tooth occasionally weakly developed or double.

Pygidium with small fleshy ventral pad and one pair of thin long lateral cirri (Fig 5D).

Glandular organs in chaetigers 5–14, largest in chaetigers 7 and 8, smallest in chaetiger 9 and gradually increasing in size from chaetiger 10 to chaetigers 12–13, slightly smaller again in chaetiger 14 (Figs 5B and 7A–7F); large organs occupying most of chaetiger space. Internal fibres long and coiled in glandular organs in chaetigers 5–8 (Fig 7B and 7C), straight, shorter and thinner in chaetigers 9–14; in fixed specimens, long straight fibers usually protruding from openings of glandular organs on chaetiger 6. Each organ on chaetigers 5, 7 and 8 opening to exterior via semicircular to suboval slit around large fiber spreader, on chaetiger 6 via small round hole, and on chaetigers 9–14 via large vertical slit. Frontal edge of fiber each spreader on chaetigers 5, 7 and 8 entire, rounded to blunt, or with variously developed middle depression vaguely separating two rounded lobes.

Digestive tract with gizzard-like structure beginning from chaetigers 15–20 and extending through 2–3 chaetigers.

Main dorsal blood vessel with heart body 10–20 μm in diameter.

Nephridia from chaetiger 15 onwards.

Methyl green staining. Intensely stained lateral sides of chaetiger 6; weakly stained lateral sides of chaetiger 5. This pattern was observed in small and large specimens from the North and Norwegian seas. In large specimens, the dorsal side of the prostomium and lateral sides of the peristomium were usually also intensely stained.

Reproduction. Spiophanes cf. bombyx is gonochoristic. The gametes proliferate in paired gonads attached to the genital blood vessels from chaetigers 18–19 to chaetigers 100–110. In females, oogenesis is intraovarian: vitellogenesis occurs when the oocytes grow in ovaries. The developed oocytes are accumulated in the coelomic cavity prior to spawning (Fig 8A). The largest intraovarian oocytes examined in fixed specimens from Norway were of irregular shape, up to 105 μm in diameter, each with a honey-combed envelope 3–4 μm thick, 45–55 cortical alveoli regularly arranged in a peripheral circle, a nucleus about 55 μm in diameter, and a single nucleolus about 20 μm in diameter. The cortical alveoli were pear-shaped, up to 8 μm long and 5 μm in diameter, with narrow necks oriented perpendicular to the oocyte surface (Fig 8B–8E).

In males, spermatogonia proliferate in testes and the rest of spermatogenesis occurs in the coelomic cavity. Spermatids are interconnected in tetrads. The spermatozoa are ect-aquasperm with a short plate-like acrosome, subspherical nucleus about 3 μm in diameter, very short midpiece, and a long flagellum (Fig 8F).

Females and males release their gametes into the water where fertilization and holopelagic, planktotrophic larval development occur. Larvae have two pairs of red eyes on the prostomium and a midventral ciliated pit on chaetiger 2 (see Hannerz [163]: Fig 9a, 9b).

Remarks. Horned Spiophanes from the North and Norwegian seas morphologically appear similar to the individual from the Adriatic Sea and almost fit the original and later descriptions of S. bombyx from the Mediterranean. All the adults have sabre chaetae and hooks in neuropodia from chaetiger 15 onwards. They differ, however, in the number of cortical alveoli which are regularly arranged in the peripheral circle in mature oocytes. Claparède ([9]: 486, pl 12, Fig 2E, 2F) noted that in worms from the Gulf of Naples the developed oocytes were lentiform (sphéroïdes aplatis), up to 130 μm in diameter, with thick papillary envelope, a large spherical germinal vesicle and a spherical nucleolus; 18 to 20 colorless cortical alveoli (“vésicules inclores” in his terminology), each about 11 μm in diameter, were arranged in a large circle. We observed 45–55 alveoli per oocyte in worms from the Norwegian Sea (Fig 8B–8E). Sequences of the North European and single Italian specimen obtained in the present study were similar (p-distances ranging from 00% for 28S to 2.46% for 16S). Nevertheless, because of the difference in the number of cortical alveoli in oocytes (which is supposed to be species specific and variable within a small range), and the interrupted distribution of the North European and the Mediterranean populations (see below S. cf. convexus), we suggest that the conspecificity of these populations should be verified in additional molecular analysis based on sequences of more genes and more individuals. Pending molecular data, we refer to the specimens from the North and Norwegian seas, and also from around Iceland and British Isles as S. cf. bombyx.

The larvae and juveniles of S. cf. bombyx were described from Frederikshavn, NW Kattegat, Denmark, by Thorson [25] and from Gullmar Fjord, Sweden, by Hannerz [163]. Hannerz [163] noted that in Gullmar Fjord 18-chaetiger juvenile about 1.3 mm long had characteristic recurved spines in neuropodia of chaetiger 1, and hooks in neuropodia from chaetiger 13.

Distribution. Northern Europe (southern limits uncertain) (Fig 4A).

Spiophanes cf. convexus Delgado-Blas et al., 2019. Figs 9 and 10A.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 9. Adult morphology of Spiophanes cf. convexus (Brittany, France, MIMB 28155).

(A, B) Formalin-fixed specimens stained with an alcohol solution of methyl green; palps missing. (A) Anterior end, left lateral view. (B) Chaetigers 5–13, left lateral view, showing specific pattern of methyl green staining. (C, D) Anterior ends, dorsal view, showing various shape of prostomium; palps missing. (E) Long thick fibers produced by glandular organ of chaetiger 6, protruding from hole-like opening of the organ. (F) Crook-like spines in neuropodium of chaetiger 1. (G) Hooded hooks from neuropodium of a middle chaetiger. (H) Posterior end, dorsal view. I, same, ventral view. Abbreviations: ch6, ch8 –chaetigers 6 and 8; fh–fronto-lateral horn; hh–hooded hook; lp–lateral peristomial lip; sa–ventral inferior sabre chaeta. Scale bars: A– 500 μm; B–D– 300 μm; E, F– 50 μm; G– 20 μm; H, I– 100 μm.

https://doi.org/10.1371/journal.pone.0234238.g009

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 10. Methyl green staining patterns in formalin-fixed specimens of Spiophanes spp.

(A) S. cf. convexus (MIMB 36704, Brittany, France), chaetigers 2–8, left lateral view. (B) S. uschakowi (MIMB 36677, Sakhalin Is., Sea of Okhotsk), chaetigers 4–7, left lateral view. (C) S. cf. norrisi (MIMB 3986, Chile), chaetigers 5–11, left lateral view. (D) S. hakaiensis n. sp. (MIMB 36709, British Columbia, Canada), chaetigers 3–7, left lateral view. (E) S. norrisi (MIMB 28153, San Diego, California, USA), chaetigers 4–8, left lateral view. Abbreviations: ch6 –chaetiger 6. Scale bars: A, B– 200 μm; C–E– 100 μm.

https://doi.org/10.1371/journal.pone.0234238.g010

?Spiophanes convexus Delgado-Blas et al. 2019 [162]: 344–347, Figs 5, 6.

Spiophanes bombyx:? Cazaux [166]: 207–210, pl 68, Figs 1–6. Meißner [8] (Part.): 54–58, Figs 33, 34. Meißner & Blank [111] (Part.): 7–11. Aylagas et al. [121]: S1 Table. Not Claparède [9, 10].

Type material. SPAIN, Galicia, Pontevedra: N° 9 Meira, 42.2833°N, 8.7167°W, Jun 76, MNCN 16.01/18442 (holotype); N° 10 Meira, 42.2833°N, 8.7167°W, Jun 76, MNCN /16.01/18444 (2 paratypes). Andalusia, Huelva, mouth of River Piedras, M.2, 37.2°N, 7.1167°W, 1988, MNCN 16.01/18443 (11 paratypes).

Adult morphology (based on our material from Brittany, France). Up to 30 mm long, 1 mm wide for 120 chaetigers. Pigmentation absent on body and palps. In formalin-fixed specimens, lateral sides of chaetigers 9–14 (until chaetigers 16–17 in large specimens) light brownish due to fixed internal content of large, probably glandular, epithelial cells.

Prostomium triangular, wide anteriorly, with a pair of distally pointed fronto-lateral horns, posteriorly narrowed, pressed into chaetiger 1 but not extending over as a caruncle. Fronto-lateral horns long (Fig 9C) or moderately developed (Fig 9D). Peristomium with narrow lateral lips closely applied to lateral sides of prostomium, and a small ventral lip. Two pairs of small dark red eyes present or eyes absent. Occipital antenna absent. Palps as long as 7–15 chaetigers.

Nuchal organs metameric, at least on 40 anterior chaetigers, fewer in small individuals. First pair of metamers long oblique ciliary bands extending from posterior part of prostomium to end of chaetiger 2; posterior ends of metamers set wider apart. Succeeding metamers shorter, each extending from nototroch over posterior half of chaetiger, oriented parallel to body axis until chaetigers 10–11, slightly oblique on succeeding chaetigers. Epithelial cells around nuchal patches with dark yellow pigment in life.

Notochaetae capillaries, long on four anterior chaetigers and on posterior chaetigers, shorter on middle chaetigers. Neuropodia of chaetiger 1 each with 1–2 heavy recurved, crook-like spines in addition to capillaries (Fig 9F). Notopodia of 5–10 posterior chaetigers each with 1–2 (usually one) long recurved spines in addition to capillaries (Fig 9H).

Sabre chaetae in neuropodia from chaetiger 15, one or rarely two in a tuft, with narrow limbation and fine granulation on distal part.

Hooks in neuropodia from chaetiger 15, up to 10 in a series, accompanied by inferior sabre chaetae throughout body and 1–2 short hair-like alimbate capillaries in 5–10 posterior chaetigers; hair-like capillaries in posterior neuropodia usually situated in upper and/or lower parts of hook row. Hooks usually quadridentate, with three small upper teeth above main fang and small subterminal hood below main fang (Fig 9G).

Nototrochs from chaetiger 1 to end of body; on each of chaetigers 1 and 2 as two short, oblique ciliary bands situated between notopodial postchaetal lamellae and nuchal ciliary bands; from chaetiger 3 through most succeeding chaetigers as complete transverse bands of dense cilia running between notopodial postchaetal lamellae. Each nototroch composed of two parallel rows of large transversally elongated cells, each bearing numerous long cilia; nototroch ciliation stronger on ridge-bearing chaetigers. Single transverse rows of smaller cells with shorter cilia present on each chaetiger beginning from chaetiger 3.

Dorsal transverse ridges present from chaetigers 14–15 to chaetigers 40–50, fewer in small individuals. Ridges thick, fleshy, one per chaetiger, not connected to notopodial lamellae. Wide blood vessel extending along each ridge internally; double nototroch with numerous long cilia arranged on top of each ridge. Intersegmental lateral pouches absent.

Pygidium with small fleshy ventral pad and one pair of thin long lateral cirri (Fig 9H and 9I).

Glandular organs in chaetigers 5–14, largest in chaetigers 7 and 8, smallest in chaetiger 9 and gradually increasing in size from chaetiger 10 to chaetigers 12–13, slightly smaller again in chaetiger 14; large organs occupying most of chaetiger space. Internal fibres long and coiled in glandular organs in chaetigers 5–8, straight, shorter and thinner in chaetigers 9–14; in fixed specimens, long straight fibers usually protruding from openings of glandular organs on chaetiger 6 (Fig 9E). Each organ on chaetigers 5, 7 and 8 opening to exterior via semicircular to suboval slit around large fiber spreader, on chaetiger 6 via small round hole, and on chaetigers 9–14 via large vertical slit (Fig 9A, 9B and 9E). Frontal edge of each fiber spreader on chaetigers 5, 7 and 8 entire, rounded to blunt, or with variously developed middle depression vaguely separating two rounded lobes.

Digestive tract with gizzard-like structure beginning from chaetigers 15–20 and extending through 2–3 chaetigers.

Main dorsal blood vessel with heart body 10–20 μm in diameter.

Nephridia from chaetiger 15 onwards.

Methyl green staining. Intensely stained lateral sides of chaetiger 6; weakly stained lateral sides of chaetigers 8–14; no staining on head or chaetigers 1–5, 7 (Figs 9A, 9B and 10A).

Remarks. Horned Spiophanes from Brittany, northern France, morphologically appear very similar or almost identical to S. bombyx from the Mediterranean and S. cf. bombyx from the North and Norwegian seas, but unambiguously differ from them in genetic characteristics (Figs 1B and 2B). They weakly differ from North European specimens also by the pattern of MG staining: no staining on head and five anterior chaetigers in S. cf. convexus, and staining on chaetiger 5 and often on the prostomium and peristomium in S. cf. bombyx. However, these patterns are variable and can only be seen in well preserved specimens.

Delgado-Blas et al. [162] described S. convexus based on the morphology of a few anterior fragments from Atlantic Spain (Galicia and Andalusia), and also from Mediterranean Spain (Catalonia). The diagnostic characters used to distinguished S. convexus from S. bombyx were ambiguous and fit into the range of morphological variability of S. bombyx, which was not examined by Delgado-Blas et al. [162]. Based on the proximity of the type locality of S. convexus (Meira, Pontevedra, Galicia) and sampling sites of specimens examined through molecular analysis in the present study, we assume that our specimens from Brittany, France, and specimens from Bay of Biscay sequenced by Aylagas et al. [121] belong to S. convexus. In the absence of sequence data of S. convexus, however, we refer our specimens to as S. cf. convexus. The identity of the horned Spiophanes from Catalonia, Spain, referred by Delgado-Blas et al. [162] to as S. convexus, should be verified in molecular analysis. So far, based on available data, we consider S. bombyx as the only horned species occurring in the Mediterranean.

To avoid confusion in identification of specimens based on morphological characters only, especially in areas of possible overlap of S. bombyx and S. convexus, and until more molecular data has been obtained from specimens from the Gulf of Naples, we suggest that North European horned Spiophanes are referred to as S. bombyx aggregate (S. bombyx agg.), rather than to a particular species.

Mesnil [11] described in detail adults and larvae of S. bombyx from Wimereux, Hauts-de-France, northern France. He noted that the adults were up to 6 cm long, 1.5 mm wide for about 180 chaetigers. They had hooks beginning invariably from chaetiger 15, whereas in larvae hooks started from chaetigers 11–14. Wimereux is located midway between the sites where S. cf. bombyx and S. cf. convexus were collected for molecular analysis. Because Mesnil’s [11] description fits both species, the identity of the worms described by him from Wimereux remains uncertain.

Cazaux [166] described in detail the larval development of a horned Spiophanes from Arcachon, France, which he identified as S. bombyx. He noted that in an early benthic 15-chaetiger juvenile, hooks were not yet developed, and in a 19-chaetiger juvenile they were present in chaetigers 13–19. Arcachon is located between sites where specimens of S. cf. convexus were collected for molecular analysis. It is plausible, therefore, that Cazaux [166] dealt with S. convexus but not S. bombyx.

Distribution. Western Europe (northern and southern limits uncertain) (Fig 4A).

Spiophanes uschakowi Zachs, 1933. Figs 10B and 11–14

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 11. Adult morphology of Spiophanes uschakowi (Peter the Great Bay, Russia, USNM 183505).

(A) Anterior end, frontal view. (B) Same, left lateral view. (C) Same, dorsal view, palps missing. (D) Same, ventral view. (E) Posterior end, left lateral view. (F) Chaetigers 7–11, dorsal view, showing longitudinal nuchal metamers, nototrochs and intersegmental transverse bands of cilia. (G) Chaetigers 12–15, dorsal view, showing oblique nuchal metamers, nototrochs and intersegmental transverse bands of cilia. (H) Palp, fronto-lateral view, showing frontal longitudinal ciliated groove. Abbreviations: ch5, ch7, ch15 –chaetigers 5, 7, 15; cr–crook-like spine in neuropodium of chaetiger 1; fh–fronto-lateral horn; lp–lateral peristomial lip; nt–nototroch; nu–nuchal organ; pa–palp scar; pr–prostomium; py–pygidial cirrus; sp–notopodial spine with recurved distal end; tc–intersegmental transverse cilia; vp–ventral peristomial lip. Scale bars: A– 50 μm; B–H– 100 μm.

https://doi.org/10.1371/journal.pone.0234238.g011

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 12. Glandular organs of Spiophanes uschakowi (Peter the Great Bay, Russia, USNM 183505).

(A) Anterior end, left lateral view. (B) Chaetigers 6–9, left lateral view, showing openings of glandular organs: small hole on chaetiger 6, suboval slits on chaetigers 7 and 8, and vertical slit on chaetiger 9. (C) Chaetigers 10–16, left lateral view, showing slit-like openings of glandular organs on chaetigers 10–14. (D) Chaetigers 4 and 5, left lateral view, showing suboval fiber spreader on neuropodium of chaetiger 5. (E) Chaetiger 6, left lateral view, showing hole-like opening of glandular organ in neuropodium. (F) Chaetigers 14 and 15, left lateral view, showing slit-like opening of glandular organ in neuropodium of chaetiger 14 and hooks in neuropodia of chaetiger 15. Abbreviations: ch5–ch16 –chaetigers 5–16; fs–fiber spreader; go–opening of glandular organ; hh–hooded hook; lo–lateral sensory ciliated organ; ne–neuropodial postchaetal lamella; no–notopodial postchaetal lamella; nt–nototroch; sa–ventral inferior sabre chaeta; se–hardened secretion protruding from opening of glandular organ; tc–intersegmental transverse cilia. Scale bars: A– 200 μm; B, C– 100 μm; D–F– 50 μm.

https://doi.org/10.1371/journal.pone.0234238.g012

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 13. Adult morphology of Spiophanes uschakowi (Peter the Great Bay, Russia, USNM 183505).

(A) Hooks in neuropodium of chaetiger 15, view from above, showing variation in number of superior teeth. (B) Same, lateral view, showing small subterminal hood below main fang. Abbreviations: fa–main fang of hook; ho–subdistal hood below main fang of hook; su–superior teeth of hook; up–upper tooth of hook. Scale bars: A, B– 2 μm.

https://doi.org/10.1371/journal.pone.0234238.g013

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 14. Glandular organs of Spiophanes uschakowi (Sakhalin Is., Sea of Okhotsk, MIMB 36677).

(A) Schematic composition of parapodia and shape of openings of glandular organs on neuropodia of chaetigers 5–9: suboval openings with one-lobe chaetal spreader on chaetigers 5, 7 and 8; small hole on chaetiger 6; and a vertical slit on chaetiger 9. (B–G) Morphology of glandular organs in chaetigers 5–12, right-side organs in left lateral view, formalin-fixed specimen stained with an alcohol solution of methyl green. (B) Chaetigers 5–12. (C) Chaetiger 5. (D) Chaetiger 7. (E) Chaetiger 8. (F) Chaetiger 9. (G) Chaetiger 10. Abbreviations: ch5–ch12 –chaetigers 5–12; fi–fibers produced by glandular organs; lo–lateral sensory ciliated organ; ne–neuropodial postchaetal lamella; no–notopodial postchaetal lamella. Scale bars: B– 200 μm; C–G– 100 μm.

https://doi.org/10.1371/journal.pone.0234238.g014

Spiophanes uschakowi Zachs, 1933 [112]: 130. Meißner [8]: 61. Meißner & Blank [111]: 15–17, Fig 7. Buzhinskaja [84]: 61. Radashevsky et al. [167]: 33–39, Figs 1–6 (gamete ultrastructure).

Spiophanes uschakovi [sic]: Annenkova [168]: 140; [113]: 172–173. Uschakov [78]: 267.

Spiophanes bombyx: Okuda [76]: 222–223, Figs 3, 4. Annenkova [113]: 172. Uschakov [77]: 200; [78]: 267; [169]: 205. Pasternak [170]: 267. Imajima & Hartman [80]: 289–290. Buzhinskaja [114]: 103; [83]: 135; [84]: 61. Koblikov [171]: 39. Ozolinsh [172]: 82–83. Imajima [88]: 128–132, Figs 8, 9; [116]: 129; [117]: 80; [118]: 379. Buzhinskaja & Britayev [115]: 85. Ozolinsh & Bagaveeva [173]: 139. Not Claparède [9, 10].

Spiophanes sp.: Radashevsky [174]: Figs 1F, 5D, 7B, 10D.

Type material. RUSSIA, Primorsky Region, Sea of Japan (East Sea), Peter the Great Bay, 42.835°N, 131.74°E, dredge, 26–30 m, fine sand, coll. K.M. Derjugin, 11 Oct 1925, ZISP 1/25826 (2 syntypes).

Adult morphology. Up to 47 mm long, 1.5 mm wide for 140 chaetigers. Pigmentation absent on body and palps. A pair of narrow transverse whitish bands, concentrations of epithelial glandular cells, visible on ventral side of chaetiger 1 in live individuals. In large formalin-fixed specimens, lateral sides of chaetigers 9–14 (until chaetigers 16–17 in large specimens) light brownish-red due to fixed internal content of large, probably glandular, epithelial cells.

Prostomium triangular, wide anteriorly, with a pair of long, distally pointed fronto-lateral horns (Fig 11A–11D), posteriorly narrowed, pressed into chaetiger 1 but not extending over it as a caruncle. Peristomium with narrow lateral lips closely applied to lateral sides of prostomium, and a small ventral lip (Fig 11B–11D). Two pairs of small dark red eyes arranged trapezoidally, comprising one pair of median eyes and one pair of lateral eyes situated anteriorly and set wider apart; occasionally eyes absent. Occipital antenna absent. Palps as long as 7–15 chaetigers, with deep frontal longitudinal groove lined with fine cilia, fronto-lateral motile compound cilia bordering frontal groove, short transverse rows of short motile compound cilia regularly arranged on inner lateral side and beating towards frontal groove, short compound non-motile cilia arising directly from palp surface and scattered on lateral and abfrontal palp surfaces (Fig 11H).

Nuchal organs metameric, at least on 25 anterior chaetigers, fewer in small individuals. First pair of metamers long oblique ciliary bands extending from posterior part of prostomium to end of chaetiger 2, shorter in small individuals; posterior ends of metamers set wider apart (Fig 11C). Succeeding metamers shorter, each extending from nototroch over posterior half of chaetiger, oriented parallel to body axis until chaetigers 9–10 (Fig 11F), oblique on succeeding chaetigers (Fig 11G).

Notochaetae capillaries, long on four anterior chaetigers and on posterior chaetigers, shorter on middle chaetigers. Neuropodia of chaetiger 1 each with 1–2 heavy recurved, crook-like spines in addition to capillaries (Figs 11A–11C and 12A). Notopodia of 5–10 posterior chaetigers each with 1–3 (usually one) long spines with recurved distal end in addition to capillaries (Fig 11E). Noto- and neuropodial postchaetal lamellae on four anterior chaetigers subtriangular, with pointed tips (Figs 11A–11D and 12A); notopodial lamellae on succeeding middle chaetigers short, subulate (Fig 11F and 11G), on posterior chaetigers long, cirriform (Fig 11E). Neuropodial postchaetal lamellae on chaetigers 5–14 large but low, rounded and fleshy, with openings of internal glandular organs (Figs 11B and 12A–12F).

Sabre chaetae in neuropodia from chaetiger 10, usually one, occasionally two per neuropodium, with fine granulation on distal part; chaetae on chaetiger 10 comparatively small, with narrow limbation, slightly reducing in size until chaetiger 14, but from chaetiger 15 onwards thicker, alimbate (Fig 12C).

Hooks in neuropodia from chaetiger 15, up to 15 in a series, accompanied by inferior sabre chaetae throughout body and 1–2 hair-like alimbate capillaries in 7–10 posterior chaetigers; hair-like capillaries in posterior neuropodia usually situated in upper and/or lower parts of hook row. Hooks usually quadridentate, with three small upper teeth above main fang and small subterminal hood below main fang (Fig 13B); median upper tooth occasionally weakly developed or doubled (Fig 13A and 13B).

Nototrochs from chaetiger 1 to end of body; on each of chaetigers 1 and 2 as two short, oblique ciliary bands situated between notopodial postchaetal lamellae and nuchal ciliary bands; from chaetiger 3 onwards as complete transverse bands of dense cilia running between notopodial postchaetal lamellae (Figs 11C, 11F, 11G and 12C). Each nototroch composed of two parallel rows of large transversally elongated cells, each bearing numerous long cilia; nototroch ciliation stronger on ridge-bearing chaetigers. Single transverse rows of smaller cells with shorter cilia present on each chaetiger beginning from chaetiger 3. These rows situated posterior to nototrochs and referred to as intersegmental transverse ciliation (Fig 11F and 11G).

Dorsal transverse ridges present from chaetiger 15 to chaetigers 30–55, fewer in small individuals. Ridges thick, fleshy, one per chaetiger, not connected to notopodial lamellae. Wide blood vessel extending along each ridge internally; double nototroch with numerous long cilia arranged on top of each ridge. Intersegmental lateral pouches absent.

Pygidium with small fleshy ventral pad and one pair of thin long lateral cirri (Fig 11E); one to two additional cirri present above lateral cirri in some individuals.

Glandular organs in chaetigers 5–14, largest in chaetigers 7 and 8, smallest in chaetiger 9 and gradually increasing in size from chaetiger 10 to chaetigers 12–13 (Fig 14B–14G), slightly smaller again in chaetiger 14; large organs occupying most of chaetiger space. Internal fibres long and coiled in glandular organs in chaetigers 5–8 (Fig 14B–14E), straight, shorter and thinner in chaetigers 9–14; in fixed specimens, long straight fibers usually protruding from openings of glandular organs on chaetiger 6. Each organ on chaetigers 5, 7 and 8 opening to exterior via semicircular to suboval slit around large fiber spreader (Figs 11B, 12A, 12B, 12D and 14A), on chaetiger 6 via small round hole (Figs 12E and 14A), and on chaetigers 9–14 via large vertical slit (Figs 12A–12C, 12F and 14A). Frontal edge of each fiber spreader on chaetigers 5, 7 and 8 entire, rounded to blunt, or with variously developed middle depression vaguely separating two rounded lobes (Fig 12A, 12B and 12D).

Foregut wide, eversible, within 3–6 anterior chaetigers, with black pigment in thick wall; oesophagus in succeeding chaetigers narrow, with thin unpigmented wall. Gizzard-like structure in end of oesophagus beginning from chaetigers 14–21 and extending through 2–3 chaetigers.

Main dorsal blood vessel with heart body extending from chaetigers 14–16 to chaetigers 20–21. Heart body soft, brownish-green, 30–40 μm in diameter. Main dorsal blood vessel transforming into gut sinus 1–3 chaetigers after gizzard-like structure. Circumoesophageal vessels joined midventrally in chaetiger 3 forming main ventral blood vessel. Blood red, without elements.

Nephridia from chaetiger 15 onwards, opening to exterior on antero-lateral edges of chaetigers in both sexes.

Habitat. Adults of S. uschakowi live in tubes in sandy to muddy-sand sediments in shallow waters. Population densities reach several thousand individuals per square meter. The tubes are sandy, firm, with thin but strong inner lining, up to 10 cm long and 2 mm in diameter, oriented vertically in sediment, with upper part 2–5 mm long protruding above the surface.

Methyl green staining. Intensely stained small area on lateral sides of chaetiger 5 (in front of openings of glandular organs; not stained in some individuals), larger area on chaetiger 6 (Fig 10B) and small fleshy pygidium (pygidial cirri not stained); weakly stained lateral sides of chaetigers 9–14.

Reproduction. Spiophanes uschakowi is gonochoristic. The gametes proliferate in paired gonads attached to the genital blood vessels from chaetigers 20–24 to chaetigers 88–120. In females, oogenesis is intraovarian: vitellogenesis occurs when the oocytes grow in ovaries. The developed oocytes are accumulated in the coelomic cavity prior to spawning. The newly released oocytes are lentiform, each 185–200 μm in diameter, with honeycombed envelope 5–7 μm thick, 41–49 cortical alveoli regularly arranged in a peripheral circle, a nucleus 80–83 μm in diameter, and a single nucleolus about 30 μm in diameter. The cortical alveoli are pear-shaped, 7–9 μm in diameter, with narrow necks oriented perpendicular to the oocyte surface (see Radashevsky et al. [167]: Fig 2).

In males, spermatogonia proliferate in testes and the rest of spermatogenesis occurs in the coelomic cavity. Spermatids are interconnected in tetrads. The spermatozoa are ect-aquasperm with a plate-like acrosome 0.58 ± 0.06 μm thick and 2.14 ± 0.13 μm in diameter, barrel-shaped nucleus 2.23 ± 0.13 μm long and 3.18 ± 0.13 μm in diameter, short midpiece 0.93 ± 0.09 μm long with five spherical mitochondria, two centrioles and one small lipid droplet, and a flagellum 62–63 μm long with 9 × 2 + 2 organization of microtubules (see Radashevsky et al. [167]: Fig 5).

Females and males release their gametes into the water where fertilization and holopelagic, planktotrophic larval development occur. Larvae have two pairs of red eyes on the prostomium and a midventral ciliated pit on chaetiger 2. Newly settled 20–21-chaetiger juveniles about 2 mm long had heavy recurved crook-like spines in neuropodia of chaetiger 1, sabre chaetae in neuropodia from chaetiger 10, hooded hooks in neuropodia from chaetiger 14, and long spines with recurved distal end in notopodia from chaetigers 17–19.

Remarks. In the original description of S. uschakowi Zachs ([112]: 130) noticed characteristic feature of worms as notopodial lamellae (“cirri dorsales”) being leaf-like on eight anterior chaetigers and cirriform on the following chaetigers. He noted that hooks appeared rather similar to those in S. bombyx and incorrectly reported that the first chaetiger had no heavy spines. Annenkova [168, 113] and Uschakov [78] likely had not seen Zachs’ material and distinguished S. bombyx and S. uschakowi only according to Zachs’ description. We have examined the two syntypes of S. uschakowi (two anterior fragments, each about 25–35 chaetigers) deposited in the polychaete collection of the Zoological Institute, Saint Petersburg, Russia (ZISP 1/25826), and observed the heavy recurved spines in first neuropodia, and other characters as they described above.

The ultrastructure of the oocytes and spermatozoa was described in details by Radashevsky et al. [167]. The larvae of the species were found in plankton collected in Peter the Great Bay, Sea of Japan (East Sea), Russia, in September-October. Their morphology will be described elsewhere.

Spiophanes uschakowi has been recorded from the Shantar Islands, northern part of Sakhalin Is. and from the South Kurile Islands south to the Korean Peninsula. Numerous records of S. bombyx from around Japanese Islands by Minoru Imajima are herein referred to S. uschakowi but the conspecificity of mainland and Japanese specimens should be verified in a further study. Remarkably, S. uschakowi has never been reported from the Chukchi Peninsula, Kamchatka Peninsula, Bering Islands, Kurile Islands, and northern part of the Sea of Okhotsk, although numerous samples from this region were provided by the expeditions of the Institute of Marine Biology, Vladivostok, FEB RAS, the Zoological Institute, St. Petersburg, RAS, some other institutions, and also during this study.

Distribution. North West Pacific: from the Sea of Okhotsk south to the Yellow Sea and East China Sea (Fig 15A).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 15. Maps showing records of Spiophanes spp. in North Pacific Ocean.

(A) Spiophanes uschakowi Zachs, 1933: red star–type locality: Peter the Great Bay, Sea of Japan (East Sea), Russia; green triangle–specimens sequenced in the present study; yellow circles–adults identified in the present study based on the morphology only; turquoise circles–adults identified as S. bombyx by Imajima [88, 116–118]. (B) Spiophanes norrisi Meißner & Blank, 2009 (yellow symbols): yellow star–type locality: Magdalena Bay, Baja California Sur, Mexico; yellow square–specimens sequenced by Meißner & Blank [111]; yellow rhombs–specimens sequenced by the LACM DISCO Project (Los Angeles, California, USA), and the Marine Biology Laboratory (City of San Diego, California, USA); yellow circles–adults identified based on the morphology only. Spiophanes hakaiensis n. sp. (green symbols): green star–type locality: Calvert Is., British Columbia, Canada; green squares–specimens sequenced in the present study; green triangles–specimens sequenced by the Canadian Centre for DNA Barcoding (Guelph, Canada), and the Marine Biology Laboratory (City of San Francisco, California, USA); green circles–adults identified based on the morphology only.

https://doi.org/10.1371/journal.pone.0234238.g015

Spiophanes norrisi Meißner & Blank, 2009. Fig 10E

Spiophanes norrisi Meißner & Blank, 2009 [111] (Part.): 11–15, Figs 5, 6A, 6B, 6D–6F.

Spiophanes bombyx: Hernández-Alcántara et al. [63]: 570. Hernández-Alcántara & Solís-Weiss [175]: 29. Méndez [176]: 142. Díaz-Castañeda & Valenzuela-Solano [177]: 513. Not Claparède [9, 10].

Type material. MEXICO, Baja California Sur, Magdalena Bay, Entrada Point, R/V Velero IV, st. 1962–50, 24.545°N, 112.06805°W, rocky intertidal with surfgrass & tide pools, coll. Allan Hancock Foundation, 3 May 1950, LACM-AHF Poly 2251 (holotype), 2254 (16 paratypes).

Synopsis. Up to 15 mm long, 0.6 mm wide for 100 chaetigers. Prostomium with long fronto-lateral horns, posteriorly pressed into chaetiger 1 but not extending over it as caruncle. Occipital antenna absent. Nuchal organs metameric, at least on 25 anterior chaetigers; first pair of metamers as oblique ciliary bands from prostomium to end of chaetiger 2; succeeding metamers shorter, parallel to body axis until chaetigers 10–11, oblique on succeeding chaetigers. Sabre chaetae in neuropodia from chaetiger 10. Hooks in neuropodia from chaetiger 15, usually quadridentate, with small subterminal hood. Pygidium with small ventral fleshy pad and one pair of lateral cirri. Glandular organs in chaetigers 5–14, largest in chaetigers 7 and 8, smallest in chaetiger 9, gradually increasing in size from chaetiger 10 to chaetigers 12–13, slightly smaller again in chaetiger 14. Internal fibres long and coiled in glandular organs in chaetigers 5–8, straight, shorter and thinner in chaetigers 9–14. Organs on chaetigers 5, 7 and 8 each opening to exterior via semicircular to suboval slit around large fiber spreader, on chaetiger 6 via small round hole, on chaetigers 9–14 via large vertical slit. Frontal edge of each fiber spreader on chaetigers 5, 7 and 8 entire, rounded to blunt, or with variously developed middle depression vaguely separating two rounded lobes. Digestive tract with gizzard-like structure. Main dorsal blood vessel with heart body. Nephridia from chaetiger 15 onwards. Gonochoristic. Oocytes lentiform, each with thick honey-combed envelope and cortical alveoli regularly arranged in a peripheral circle. Spermatids interconnected in tetrads. Spermatozoa short-headed aqua-sperm with plate-like acrosomes. Fertilization in sea water. Larval development holopelagic, planktotrophic. Larvae with two pairs of red eyes on prostomium and a midventral ciliated pit on chaetiger 2.

Remarks. Meißner & Blank [111] designated 17 specimens from one sample collected from Magdalena Bay, Baja California Sur, Pacific Mexico, as the type material of S. norrisi (Fig 15B). They referred to numerous specimens of horned Spiophanes collected along the Pacific American coast from Alaska south to Chile as non-type material of the same species. For molecular analysis (short fragments of COI), the authors used specimens collected from offshore San Francisco (SF), northern California.

Molecular analysis performed in the present study suggested the presence of two sibling vicariant species distributed along the Pacific coast of North America and overlapping in California (Figs 2B and 3). Herein, we refer northern specimens to a new species: Spiophanes hakaiensis n. sp. (see below). The COI sequences provided by Meißner & Blank [111] were grouped together with sequences from specimens from off San Francisco and Baja California Norte, Mexico, provided in other studies (see Table 1, Fig 2B). Consequently, these specimens are referred to as S. norrisi. South American populations of horned Spiophanes have not been examined in genetic analysis. Pending molecular data, we suggest that Chilean and Argentinean specimens are referred to as S. cf. norrisi (see below).

Meißner & Blank [111] noticed that S. norrisi differed from morphologically similar species mainly in the orientation of segmental nuchal metamers (“dorsal ciliated patches”, “metameric ciliated patches” or “dorsal ciliated organs” in their terminology), and that it was the only known species with oblique orientation of the metamers from as early as between chaetigers 9–10. After describing horned American Spiophanes as a new species, S. norrisi, Meißner & Blank ([111]: 6) noted that S. uschakowi “turned out” to be very similar to S. norrisi, but “a definite assignment [of S. uschakowi] based on morphological characters was not possible as long as the nature of the dorsal ciliated organs posterior to chaetiger 2 was unknown.” We observed segmental nuchal metamers oriented parallel to body axis until chaetigers 9–10 in S. uschakowi and until chaetigers 10–11 in S. norrisi; thus the two species cannot be distinguished based on the morphology of their nuchal organs. We did not observe sabre chaetae beginning earlier than chaetiger 10 in S. norrisi and interpret their rare start from chaetiger 9 reported by Meißner & Blank [111] as an abnormality, as with the occasional bifurcation of one or both pygidial cirri rarely occurring both in S. uschakowi and S. norrisi. Individuals of both species demonstrate the same variability in the shape of the chaetal spreaders on chaetigers 5, 7 and 8, and all other morphological characters examined so far. Specimens of the two species differ slightly in the patterns of the MG staining. In S. uschakowi, the lateral sides of chaetiger 5 (in front of openings of glandular organs) are usually weakly stained (Fig 10B), whereas in S. norrisi such staining has not been observed (Fig 10E). The staining pattern in S. uschakowi, however, is variable and in some individuals (worms from Peter the Great Bay were mostly examined on this account) staining was not revealed on chaetiger 5, making this character also unreliable for distinguishing the two species.

Blake [178] described 13- and 14-chaetiger horned larvae of a Spiophanes collected in Tomales Bay, California, in November 1971. The 14-chaetiger larva was 1025 μm long and had hooded hooks in neuropodia from chaetiger 11. Blake [178] noticed subtle morphological differences between the Californian and Swedish larvae (described by Hannerz [163]) and referred the former to as S. cf. bombyx. However, two other horned Spiophanes, S. anoculata Hartman, 1960 and S. hakaiensis n. sp., also occur in Californian waters. As with S. bombyx and S. norrisi, adults of S. anoculata have hooded hooks in neuropodia from chaetiger 15. So far, S. anoculata has only been found in deep water, from 922 m to 2800 m depth (see Meißner [8]), but because Blake [178] collected very few (only two) larvae of same kind in shallow-water plankton, these larvae may belong to a deep-water species. Thus, their identity remains uncertain.

Distribution. North East Pacific, California: from San Francisco Bay, USA, south to Baja California Sur, Mexico (Fig 15B).

Spiophanes hakaiensis Radashevsky & Pankova, n. sp.. urn:lsid:zoobank.org:act:20552AB1-C5D1-4310-A8B2-5BCE4370ECFA

Figs 10D and 16

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 16. Juvenile and adult morphology of Spiophanes hakaiensis n. sp.

(A–E)–live 24-chaetiger juvenile. (A) Complete individual. (B) Anterior end and pygidium, dorsal view. (C) Right palp, frontal view. (D) Posterior chaetigers, left lateral view. (E) Gizzard-like structure, dorsal view. (F) Anterior end of a live adult individual, dorsal view. (G) Left neuropodium of chaetiger 6 of a formalin-fixed 40-chaetigers juvenile, ventral view, showing short neurochaetae and long thin straight fibers protruding from hole-like opening of a glandular organ. Abbreviations: ch5–ch10 –chaetiger 5–10; ci–transverse rows of short motile compound cilia on inner lateral side of palp; dv–main dorsal blood vessel; fg–foregut; fh–fronto-lateral horn; fi–fibers produced by glandular organs; gi–gizzard-like structure; go–glandular organs; mg–midgut; ne–capillary neurochaetae; oe–oesophagus; pg–posterior gut; py–pygidial cirri; sp–notopodial spines with recurved distal end. Scale bars: A, B– 100 μm; C–E, G– 50 μm; F– 300 μm. A–E–British Columbia, Canada, MIMB 36708, F–British Columbia, Canada, CMNA 2019–0105 (holotype). G–Oregon, USA, MIMB 36711.

https://doi.org/10.1371/journal.pone.0234238.g016

Spiophanes norrisi: Meißner & Blank, 2009 [111] (Part.): 11–15.

Spiophanes bombyx: Berkeley [53]: 416; [179]: 560. Berkeley & Berkeley [57]: 22–24, Figs 40–43. Hobson & Banse [62]: 45, Fig 6l. Carey [180]: 438. Macdonald et al. [181]: 40. Carr [46]: Supplement. Not Claparède [9, 10].

Type material. CANADA, British Columbia, 2017 Hakai-MarineGEO BioBlitz, Queen Charlotte Sound, Calvert Is.: Choked Pass, North Pigu Is., st. IHAK-46B, 51.6763°N, 128.1222°W, 6 m, sea grass, coll. D. VanMaanen, 4 Aug 2017, CMNA 2019–0105 (holotype); Choked Pass, st. IHAK-42B, 51.6806°N, 128.1163°W, 5 m, outer sandspit sediment, coll. G. Paulay, 3 Aug 2017, MIMB 36708 (paratype); Pruth Bay, st. SHAK-10, 51.6441°N, 128.1195°W, low intertidal, muddy sand, eel grass, coll. S. Dudas, 24 Jul 2017, MIMB 36707 (paratype). USA, California, offshore San Francisco: st. SWOO-63, 37.6588°N, 122.5615°W, 27.5 m, coll. CCSF/SFPUC Lab, 13 Sep 2010, MIMB 39021 (13 paratypes); st. SWOO-73, 37.7125°N, 122.5648°W, 18.5 m, muddy sand, 13 Sep 2010, MIMB 39022 (2 paratypes).

Adult morphology (based on the type material–specimens identified by means of molecular analysis in the present study). Up to 25 mm long, 1.3 mm wide for 110 chaetigers. Pigmentation absent on body. Few examined live complete individuals with fine black pigment scattered on palps (Fig 16F). In large formalin-fixed specimens, lateral sides of chaetigers 9–14 (until chaetigers 16–17 in largest specimens) light reddish due to fixed internal content of large, probably glandular, epithelial cells.

Prostomium triangular, wide anteriorly, with a pair of long, distally pointed fronto-lateral horns (Fig 16A, 16B and 16F), posteriorly narrowed, pressed into chaetiger 1 but not extending over as a caruncle. Peristomium with narrow lateral lips closely applied to lateral sides of prostomium, and a small ventral lip. Two pairs of small dark red eyes arranged trapezoidally, comprising one pair of median eyes and one pair of lateral eyes situated anteriorly and set wider apart; occasionally eyes absent. Occipital antenna absent. Palps as long as 7–15 chaetigers, with deep frontal longitudinal groove lined with fine cilia, fronto-lateral motile compound cilia bordering frontal groove, short transverse rows of short motile compound cilia regularly arranged on inner lateral side and beating towards frontal groove (Fig 16C and 16F), short compound non-motile cilia arising directly from palp surface and scattered on lateral and abfrontal palp surfaces.

Nuchal organs metameric, at least on 25 anterior chaetigers, fewer in small individuals. First pair of metamers long oblique ciliary bands extending from posterior part of prostomium to end of chaetiger 2, shorter in small individuals; posterior ends of metamers set wider apart. Succeeding metamers shorter, each extending from nototroch over posterior half of chaetiger, oriented parallel to body axis until chaetigers 10–11, oblique on succeeding chaetigers.

Notochaetae capillaries, long on four anterior chaetigers and on posterior chaetigers, shorter on middle chaetigers. Neuropodia of chaetiger 1 each with 1–2 heavy recurved, crook-like spines in addition to capillaries. Notopodia of 5–10 posterior chaetigers each with 1–3 (usually one) long spines with recurved distal end in addition to capillaries (Fig 16D). Noto- and neuropodial postchaetal lamellae on four anterior chaetigers subtriangular, with pointed tips; notopodial lamellae on succeeding middle chaetigers short, subulate, on posterior chaetigers long, cirriform. Neuropodial postchaetal lamellae on chaetigers 5–14 large but low, rounded and fleshy, with openings of internal glandular organs.

Sabre chaetae in neuropodia from chaetiger 10, usually one, occasionally two per neuropodium, with fine granulation on distal part; chaetae on chaetiger 10 comparatively small, with narrow limbation, slightly reducing in size until chaetiger 14, but from chaetiger 15 onwards thicker, alimbate.

Hooks in neuropodia from chaetiger 15, up to 10 in a series, accompanied by inferior sabre chaetae throughout body and 1–2 hair-like alimbate capillaries in 5–10 posterior chaetigers. Hooks usually quadridentate, with three small upper teeth above main fang and small subterminal hood below main fang.

Nototrochs from chaetiger 1 to end of body, each composed of two parallel rows of large transversally elongated cells, each bearing numerous long cilia; nototroch ciliation stronger on ridge-bearing chaetigers. Intersegmental transverse ciliation from chaetiger 3 onwards.

Dorsal transverse ridges from chaetigers 14–15 to chaetigers 30–55, fewer in small individuals. Intersegmental lateral pouches absent.

Pygidium with small fleshy ventral pad and one pair of thin long lateral cirri.

Glandular organs in chaetigers 5–14, largest in chaetigers 7 and 8, smallest in chaetiger 9 and gradually increasing in size from chaetiger 10 to chaetigers 12–13, slightly smaller again in chaetiger 14; large organs occupying most of chaetiger space. Internal fibres long and coiled in glandular organs in chaetigers 5–8, straight, shorter and thinner in chaetigers 9–14; in fixed specimens, long straight fibers usually protruding from openings of glandular organs on chaetiger 6 (Fig 16G). Each organ on chaetigers 5, 7 and 8 opening to exterior via semicircular to suboval slit around large fiber spreader, on chaetiger 6 via small round hole, and on chaetigers 9–14 via large vertical slit. Frontal edge of each fiber spreader on chaetigers 5, 7 and 8 blunt or with variously developed middle depression vaguely separating two rounded lobes.

Foregut wide, eversible, within 3–4 anterior chaetigers, with greenish-black pigment in thick wall; oesophagus in succeeding chaetigers narrow, with thin unpigmented wall (Fig 16F). Gizzard-like structure in end of oesophagus beginning from chaetigers 10–20 and extending through 2–3 chaetigers (Fig 16B–16E). Posterior gut with transparent wall.

Main dorsal blood vessel with heart body extending from chaetigers 12–15 to chaetigers 19–20. Blood red, without elements (Fig 16F).

Nephridia from chaetiger 15 onwards, opening to exterior on antero-lateral edges of chaetigers in both sexes.

Juvenile morphology. 23-chaetiger juvenile about 2 mm long, 0.3 mm wide on chaetiger 5, with fronto-lateral horns on prostomium well developed (Fig 16A). First pair of nuchal metamers extending to middle of chaetiger 2. Succeeding metamers on chaetigers 6–11. Transverse rows of short motile compound cilia regularly arranged on distal half of each palp (Fig 16C). Double-row nototrochs from chaetiger 3 onwards. Sabre chaetae in neuropodia from chaetiger 11, very small on the first chaetiger, better discernible from chaetiger 12. Hooks in neuropodial from chaetiger 14. Long spines with recurved distal end in notopodia of chaetigers 19–23 (Fig 16D). Pygidium with ventral semispherical yellow pad and one pair of thin transparent dorsal cirri (Fig 16A and 16B). Gizzard-like structure in chaetigers 10–11. Nephridia from chaetiger 15 onwards.

30–40-chaetiger juveniles about 0.4 mm wide with sabre chaetae in neuropodia from chaetiger 10, 1–2 hooded hooks among 4–6 capillary chaetae and single sabre chaeta in each neuropodium of chaetiger 14, and only hooks and sabre chaetae in neuropodia from chaetiger 15 onwards. Individuals more than 0.4 mm wide with morphological characters as described above in Adult morphology, with sabre chaetae in neuropodia from chaetiger 10 and hooded hooks from chaetiger 15.

Methyl green staining. Intensely stained lateral sides of chaetiger 6 and small fleshy pygidium (pygidial cirri not stained), weakly stained lateral sides of chaetigers 9–14; no staining on chaetigers 1–5 and 7, 8 (Fig 10D).

Reproduction. Spiophanes hakaiensis n. sp. is gonochoristic. Mature females and males collected in British Columbia, Canada, in May 1966 (RBCM 55–47), have gametes from chaetiger 21 through most of the body. The oocytes have honey-combed envelopes with cortical alveoli regularly arranged in a peripheral circle. Spermatids are interconnected in tetrads; the spermatozoa were ect-aquasperm with a short acrosome and short, subspherical nucleus about 3 μm in diameter.

Remarks. Horned Spiophanes from the North East Pacific appear morphologically very similar or almost identical to each other and to S. uschakowi from the North West Pacific. However, molecular analysis performed in the present study showed significant difference between the North East and North West populations and also suggested the presence of two sibling vicariant species distributed along the Pacific coast of North America and overlapping in California (Figs 1B, 2B and 3). Sequences of some Californian specimens (including COI sequences provided by Meißner & Blank [111]) were grouped together with sequences of specimens from Baja California Norte, Mexico (see Table 1, Fig 1B). Because the latter locality is close to the type locality of S. norrisi, those specimens are referred to this species. Other Californian specimens were genetically similar to horned Spiophanes from Alaska, USA, and British Columbia, Canada. Those specimens are herein referred to a new species: Spiophanes hakaiensis n. sp. Comments on the taxonomy and the distribution of these species are provided above in the Remarks for S. norrisi, and below in the Remarks for S. cf. norrisi.

To avoid confusion in the identification of specimens based on the morphology only, in areas of overlap of S. hakaiensis n. sp. and S. norrisi, we suggest that specimens from California between Point Reyes (38°N) and Point Conception (34.45°N) are referred to as S. norrisi aggregate; specimens occurring from Point Reyes north to Alaska are herein referred to as S. hakaiensis n. sp.; whereas those occurring from Point Conception south to Baja California Sur, Mexico, are referred to as S. norrisi.

Distribution. From Alaska south to San Francisco Bay, California, USA (Fig 15A).

Spiophanes cf. norrisi Meißner & Blank, 2009. Fig 10C

Spiophanes bombyx: Hartman [109]: 22, pl V, Figs 14–16. Carrasco [67]: 197–199, Figs 37–41; [119]: 48–53, Figs 19, 20, 22 J–L (larval morphology). Blake [48]: 230. Rozbaczylo & Salgado [71]: 23, Fig 2e. Carrasco [182]: 453, 455. Mendez et al. [183]: 431. Montiel et al. [73]: 310. Not Claparède [9, 10].

Spiophanes chilensis: Hartmann-Schröder [184] (Part.): 215–218.

Spiophanes norrisi: Meißner & Blank [111] (Part.): 11–15, Fig 6C.

Remarks. Hartmann-Schröder [184] described Chilean Spiophanes (distributed from Punta Tortuga, Coquimbo, south to Gulf of Corcovado) with prostomium expanded anteriorly as S. chilensis Hartmann-Schröder, 1965. Meißner [8] and Meißner & Blank [111] re-examined the type material of S. chilensis and distinguished specimens belonging to three species: Spiophanes duplex (Chamberlin, 1919), Spiophanes fimbriata Moore, 1923, and S. norrisi. Meißner [8] referred the holotype of S. chilensis to S. duplex and considered the former species as a junior synonym of the latter (see below). Meißner & Blank ([111]: 14–15) referred 22 paratypes of S. chilensis from Punta Tortuga (29°57.350' S, ZMH P-14946) and additional material from Bahia Quillaipe (41°32.469' S, ZMH P-21118, 2 spec.) to S. norrisi and concluded that the latter species “occurs from shallow waters up to subtidal depths waters along the North and South American coast.”

Meißner & Blank [111] did not mention the records of S. bombyx from Chile by Carrasco [67, 119] and from Argentina and the Falkland Islands by Blake [48], although these two authors noticed that their specimens were identical with S. bombyx from Europe. We also had no chance to examine Carrasco’s or Blake’s material but got new material from Chile and Argentina (see S1 Table). We could not find any difference between this material and corresponding specimens from the Pacific USA and Canada, either in the morphology or in the MG staining (Fig 10C–10E). Nevertheless, pending results of a comparative molecular analysis of North and South American specimens (see above Remarks for S. norrisi), we suggest that Chilean and Argentinean specimens are referred to as S. cf. norrisi. It is quite possible that South American horned Spiophanes, although morphologically appearing similar to North American S. norrisi, differ from them genetically and represent a different species. We see support to this idea in the limited distribution of worms in the southern part of South America only.

Distribution. South America: Chile, Argentina, Falkland Islands (Fig 17).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 17. Map showing type localities and records of Spiophanes spp. in North and South Americas.

https://doi.org/10.1371/journal.pone.0234238.g017

Spiophanes duplex (Chamberlin, 1919) (green and yellow symbols): green star–type locality: Laguna Beach, California, USA; green triangle–specimens sequenced in the present study; yellow star–type locality of Spiophanes missionensis Hartman, 1941 (junior synonym of S. duplex): Mission Bay, California, USA. Spiophanes soederstroemi Hartman, 1953 (red and pink symbols): red star–type locality: off Rio Grande do Sul, Brazil; red triangle–specimens sequenced in the present study; pink star–type locality of Spiophanes chilensis Hartmann-Schröder, 1965 (junior synonym of S. soederstroemi): Punta Tortuga near Coquimbo, Chile. Spiophanes cf. norrisi: turquoise circles–reports as S. bombyx by Blake [48], Mendez et al. [183], Meißner & Blank [111], and identified in the present study based on the morphology only.

Spiophanes duplex (Chamberlin, 1919)

Morants duplex Chamberlin, 1919 [185]: 17. Fide Blake [64]: 149.

Spiophanes missionensis Hartman, 1941 [186]: 296–298, pl 46, Figs 17–21; [56]: 46; [187]: 46. Reish & Winter [188]: 113. Barnard & Reish [189]: 88. Reish & Barnard [190]: 5. Reish [191]: 84; [192]: 77. Kauwling & Reish [193]: 58. Hernández-Alcántara et al. [63]: 570. Maurer et al. [194]: 185–203. Méndez [176]: 142. Salazar-Vallejo & Londoño-Mesa [195]: 61. Fide Blake [64]: 149.

Spiophanes duplex: Blake [64]: 149–150, Fig 4.20; [178]: 592–593, Fig 13.11 (larval morphology). Hernández-Alcántara & Solís-Weiss [175]: 29; [196]: 285. Salazar-Vallejo & Londoño-Mesa [195]: 61. Meißner [8] (Part.): 31–36. Blake & Ruff [197]: 377. Díaz-Castañeda & Valenzuela-Solano [177]: 513. Schiff et al. [198]: 41–42.

Type material. USA, California, Orange Co., Laguna Beach, Balboa, 33.5419°N, 117.7892°W, intertidal, MCZ ANNb-2165 (holotype).

Remarks. Spiophanes duplex was originally described from Laguna Beach, Balboa, California, USA, by Chamberlin ([185], as Morants duplex). The original description was brief, with some features misinterpreted, without any illustration. Misinterpreted features greatly distinguished these worms from other spionids, and based on them, Chamberlin [185] established a new genus, Morants Chamberlin, 1919. In the Catalogue of the Polychaetous Annelids of the World, Hartman [1999] noted Morants as a monotypic genus. The genus and the species have been enigma for many years until Blake [64] discovered the holotype of M. duplex in the collections of the Museum of Comparative Zoology at Harvard (MCZ 2165) and redescribed it. He noted that the holotype had all the characters of Spiophanes and referred to Morants as a junior synonym of Spiophanes. Blake [64] also referred to Spiophanes missionensis Hartman, 1941, originally described from Mission Bay, San Diego County, California, USA, as a junior synonym of S. duplex.

Meißner [8] reexamined the type specimens of S. missionensis Hartman, 1941, S. soederstroemi Hartman, 1953, and holotype of S. chilensis Hartmann-Schröder, 1965 and, based on their morphological similarity, considered these species as junior synonyms of S. duplex (Chamberlin, 1919). Consequently, she suggested that S. duplex is widely distributed along both the Pacific and Atlantic coasts of both North and South America.

The results of the present study unambiguously show that specimens from Brazil, being morphologically identical to adults of S. duplex from California, differ from them essentially in genetic characteristics (Fig 2B). Therefore, we considered the two populations as not conspecific and refer the Brazilian population to S. soederstroemi (see below). Pending results of molecular studies in other American regions, we consider S. duplex as distributed in California only.

Distribution. North East Pacific: California (Fig 17).

Spiophanes soederstroemi Hartman, 1953. Spiophanes kroyeri: Söderström [21] (Part.): 243. Not Grube [200]. Fide Hartman [201]: 41.

Spiophanes soederstroemi Hartman, 1953 [201]: 41, Fig 14. Orensanz & Gianuca [202]: 17–18. Blake [48]: 230–232, Fig 13. Rozbaczylo & Castilla [203]: 175. Rozbaczylo & Salgado [71]: 24–25, Fig 2f, g. Carrasco [182]: 453, 455. Cañete et al. [204]: 247. Maciolek [205] (Part.): 543–544. Lancellotti & Stotz [206]: 309. Palma et al. [207]: 240. Montiel et al. [73]: 310.

Spiophanes chilensis Hartmann-Schröder, 1965 [184] (Part.): 215–218, Figs 208–211. Fide Blake [48]: 230.

Spiophanes missionensis: Bolivar & Lana [49]: 132–134, Figs 48–53; [50]: 255. Morgado & Amaral [208]: 549. Paiva [209]: 42; [210]: 74. Muniz & Pires [211]: 520; [51]: 152. Arasaki et al. [212]: 258. Santi & Tavares [213]: 293. Not Hartman [186].

Spiophanes duplex: Meißner [8] (Part.): 31–36, Figs 16–19. Pardo et al. [52]: 220, textfigs A–F. Scarabino [214]: 121. Pagliosa et al. [215]: 46. Radashevsky [174]: Fig 12A. Shimabukuro et al. [216]: 9. Not Chamberlin [185].

Spiophanes sp.: Radashevsky [174]: Figs 4A, 10C, 10D.

Type material. Off Brazil, Rio Grande do Sul, Swedish Antarctic Expedition 1901–1903, st. 1, 33.00°S, 51.1667°W, 80 m, bottom of dark gray mud, 12 Dec 1901, SMNH 5369 (2 syntypes).

Remarks. Spiophanes soederstroemi Hartman, 1953 was established by Hartman [201] based on two specimens from South America collected by the Swedish Antarctic Expedition 1901–1903 onboard the ship Antarctic. The specimens were first reported by Söderström [21] as a new record for S. kroyeri. Remarkably, Söderström ([21]: 243) and all the following authors noted that the specimens were collected on December 12, 1901 at station 1, off Uruguay. However, the coordinates provided for this station by Söderström [21] and also in a copy of the actual station list made during the expedition: 33°00'S, 51°10'W (SMNH, Lena Gustavsson in litt., 11 Sep 2019), and a map of the Expedition route with daily positions of the ship [217] show a sampling site of December 12, 1901 situated off Rio Grande do Sul, Brazil (Fig 17), north of the border between Brazil and Uruguay. Noteworthy, although this border was already well established in the mid 19^th century and did not change after that, it was not shown on the Expedition map. Possible uncertainty in its exact position might create confusion about reference on the type locality of S. soederstroemi.

Blake [48] redescribed two syntypes of S. soederstroemi, reviewed a complicated history of this species and clarified some of its morphological features and taxonomic issues. He also reexamined the type specimens of Spiophanes chilensis Hartmann-Schröder, 1965, which was originally described from Punta Tortuga, Chile, by Hartmann-Schröder [184], and referred its holotype (ZMH P-14946) to S. soederstroemi. Consequently, Blake [48] considered S. chilensis as a junior synonym of S. soederstroemi. He also noticed subtle differences between S. soederstroemi and S. missionensis and considered them as two closely related but separate species (the holotype of Morants duplex was not yet discovered at that time).

Meißner [8] reexamined type specimens of S. missionensis Hartman, 1941, S. soederstroemi Hartman, 1953, and holotype of S. chilensis Hartmann-Schröder, 1965 and, based on their morphological similarity, considered these species as junior synonyms of S. duplex (Chamberlin, 1919).

During the present study, we examined live Spiophanes in California, USA, and Paraná, Brazil, which fit all the characteristics of S. duplex. We could not find any reliable morphological difference between them, but the differences between their molecular characteristics suggest the presence of two distinct species (Fig 2B). Consequently, we treat S. duplex and S. soederstroemi as two valid species, and consider S. missionensis as a junior synonym of S. duplex, and S. chilensis as a junior synonym of S. soederstroemi. The type localities of these species and sampling sites for the specimens used in the present molecular analysis are shown on Fig 17.

Spiophanes soederstroemi is likely distributed in South America only. Records of S. soederstroemi from South Africa [100, 101], South Georgia [109], Antarctica [218, 219], China [90], Florida and North Carolina, USA [205], Philippines [220], and the Arabian Gulf [221] possibly represent misidentifications of different species.

Distribution. South America: Brazil, Uruguay, Argentina, Chile (Fig 17).

Spiophanes kroyeri Grube, 1860. Spiophanes kroyeri Grube, 1860 [200]: 88–89, pl V, Fig 1. Sikorski [33] (Part.): 329–331, textfig Meißner [8] (Part.): 7–14, Fig 1A.

Type material. Greenland Sea, off northern Iceland, coll. Grube, E., ZMB Q4746 (lectotype), 11168 (paralectotype).

Remarks. Grube ([200]: 89) noted in the original description of S. kroyeri that the new species was “Aus dem Meere von Grönland.” No more detail about the type locality has ever been published, but the original registry catalogue and labels for the types of S. kroyeri maintained in the polychaete collection of the Zoologisches Museum Berlin (ZMB), Germany, say that the material was from Iceland, i.e. from the southern part of the Greenland Sea ([222]; Birger Neuhaus in litt., 15 Jul 2019). A tentative position of the type locality is shown on the Fig 4B.

Meißner ([8]: 13) considered S. kroyeri as “the most problematic species within the genus.” She reviewed its worldwide reports and suggested that most of them were based on misidentifications. To clarify the identity of S. kroyeri, Meißner [8] designated a lectotype (ZMB Q4746) and provided a series of diagnostic characteristics of the adults. They included a bell-shaped prostomium, well developed occipital antenna, nuchal organs as a pair of longitudinal ciliary bands extending to chaetigers 14–16, suboval chaetal spreaders well developed on chaetigers 5–7, sabre chaetae in neuropodia from chaetiger 4, hooks with subterminal hood in neuropodia from chaetiger 15, lateral pouches first appearing between neuropodia of chaetigers 15 and 16, and pigmentation comprising light brown pigment in postchaetal lamellae of chaetigers 1–7 and brown pigment in neuropodia of chaetigers 7–14, most conspicuous in chaetigers 8–11.

Meißner ([8]: 14) commented on the problems with the original description and type material of S. kroyeri reyssi described from Mediterranean France by Laubier [223]. She raised it to species level, S. reyssi Laubier, 1964, and emphasized that “so far only specimens from the North Atlantic can be assigned to S. kroyeri”.

The analysis of COI sequences of Spiophanes from the North Sea obtained by Meißner & Blank [111], from the Bay of Biscay, Spain, obtained by Aylagas et al. [121], and from the Barents Sea, Norway, obtained in the present study (see Table 1), revealed two distinct groups among specimens identified by morphology as S. kroyeri (Fig 1B). Worms of one group occurred in the Barents Sea, Norway, and in the northern part of the North Sea (vouchers HH64, 94 by Meißner & Blank [111]), whereas worms of another group occurred in the northern (vouchers HH18, 19 by Meißner & Blank [111]) and central parts of the North Sea (voucher HH63 by Meißner & Blank [111]), and in the Bay of Biscay, Spain. According to their distributions, we call these groups northern and southern, respectively. In relation to the type localities of S. kroyeri (Greenland Sea, off northern Iceland) and S. cirrata (Oslofjord, southern Norway, see below), we tentatively refer to the northern-group specimens as S. cf. kroyeri, and to the southern-group specimens as S. cf. cirrata (Fig 4B). Final conclusion about the specific identity of these worms can be inferred after molecular examination of worms from the type localities of the corresponding species in Iceland, Norway, and Mediterranean France. To avoid confusion in the identification of S. kroyeri-like specimens from the northern part of the North Sea (area of overlap of the two species) based on morphological characters only, we suggest that they are referred to as S. kroyeri aggregate (S. kroyeri agg.), rather than to a particular species.

It is noteworthy that deep-water Spiophanes worms from off the Crozet Islands, Southern Ocean, referred to as S. kroyeri by Mincks et al. [122], are genetically different from European specimens (Fig 2B) and are therefore referred to herein as S. aff. kroyeri.

Distribution. Arctic; North Atlantic (Fig 4B).

Spiophanes cirrata M. Sars in G.O. Sars, 1872. Spiophanes cirrata M. Sars in G.O. Sars, 1872 [224]: 410–411. M. Sars [225]: 268–273, pl XVIII, Figs 1–16.

?Spiophanes kroyeri: Hannerz [163]: 36–40, Figs 10, 11 (larval morphology). Eliason [19]: 49–50; [20]: 263. Hartmann-Schröder [164]: 326–327, Fig 111; [165]: 342–343, Fig 157. Böggemann [32]: 121, Fig 98. Jelsing [226]: 244, 247, Fig 1I (nuchal organs). Meißner [8] (Part.): 7–14, Figs 1B–H, 2, 3. Zettler et al. [227]: checklist. Not Grube [200].

Not Spiophanes cirrata: Berkeley [53]: 416; [179]: 560. Berkeley & Berkeley [228]: 475, textfig; [57]: 24–25, Figs 44–46; [229]: 791. Carey [180]: 439.

Type material. NORWAY, North Sea, Skagerrak, Oslofjord, Drøbak, types missing, Oslo, Norway.

Remarks. Michael Sars (in G.O. Sars [224]) briefly described Spiophanes cirrata based on material from Drøbak, Oslofjord, southern Norway (Fig 4B), and Skrova, small island group in the Lofoten Archipelago, northern Norway. Later, Sars [225] illustrated and provided a more complete re-description of this species. Tauber [230] and Söderström [21] considered S. cirrata to be a junior synonym of S. kroyeri. Hartman [199] listed S. cirrata as a valid species. Hartmann-Schröder [164, 165] and Kirkegaard [231] neither noted nor commented on this species. As the first sampling site mentioned in the original description of the species by Michael Sars, Drøbak in Oslofjord is herein for the first time recognized as the type locality of Spiophanes cirrata.

Based on the results of the molecular analysis performed in the present study (see above Remarks for Spiophanes kroyeri), and according to the type localities of S. kroyeri and S. cirrata, we tentatively refer S. kroyeri-like worms from the Barents Sea, Norway, and the northern part of the North Sea to as S. cf. kroyeri, and those from the northern and central parts of the North Sea, and from the Bay of Biscay, Spain, to as S. cf. cirrata (Fig 4B). Final conclusions about the specific identity of these worms can only be inferred after molecular examination of worms from the corresponding type localities of these species.

Distribution. North East Atlantic (Fig 4B).

Spiophanes berkeleyorum Pettibone, 1962. Spiophanes berkeleyorum Pettibone, 1962 [232]: 78–83, Figs 1–4. Light [60]: 78–79, Fig 5a, b; [61]: 63–66, textfigs 63–65. Hobson & Banse [62]: 45, Fig 6k. Blake [64]: 143–145, Fig 4.17. Meißner [8]: 28–31, Figs 13–15. Blake & Ruff [197]: 377. Carr [46]: Supplement.

Spiophanes cirrata: Berkeley [53]: 416; [179]: 560. Berkeley & Berkeley [228]: 475, textfig; [57]: 24–25, Figs 44–46; [229]: 791. Carey [180]: 439. Not M. Sars in G.O. Sars [224]. Fide Pettibone [232].

Type material. CANADA, British Columbia, east coast of Vancouver Island, Departure Bay beach, coll. E. & C. Berkeley, 25 Apr 1936, USNM 30399 (holotype), 30400 (6 paratypes).

Remarks. Spiophanes berkeleyorum was originally described from Vancouver Island, British Columbia, Canada, by Pettibone [232] and later reported from distant regions. Meißner & Hutchings [110] removed Spiophanes japonicum Imajima, 1991 from synonymy with S. berkeleyorum and Meißner [8] suggested that the latter species is distributed along the Pacific coast of North America only. Later reports of the species from Brazil [233, 215], Russia [84], and China [95] probably represent misidentifications and should be verified in additional studies. Molecular data of S. berkeleyorum are limited to sequences provided by Meißner & Blank [111] and those in the present study.

Distribution. North East Pacific: from Alaska south to California.

Trochochaeta multisetosa (Örsted, 1843). Disoma multisetosum Örsted, 1843 [234]: 41–42; [235]: 107–108, pl II, Figs 1–12. Thulin [236]: 9, Figs 7–17. Ditlevsen [237]: 32. Friedrich [23]: 135, Fig 88. Wesenberg-Lund [238]: 31, pl 7, Figs 31–32. Hannerz [163]: 141, Figs 51–52. Fide Pettibone [239]: 310.

Trochochaeta multisetosa: Pettibone [239]: 310–315, Figs 82, 83 a–g.

Trochochaeta multisetosum: Weitbrecht [240]: 401–412, Figs 1–10.

Disoma franciscanum Hartman, 1947 [241]: 160–169, Figs 1–3. Fide Pettibone [239]: 310.

Trochochaeta franciscanum: Blake & Arnofsky [242]: 68, Fig 6. Blake [178]: 598–600, Fig 13.13. Blake & Ruff [197]: 377.

Trochochaeta franciscana: Radashevsky et al. [243]: 574–575.

Type material. SWEDEN, Near Hveen (Ven) Island in the Øresund Strait. Types missing.

Remarks. Although Trochochaeta was not an object of the present study, use of these worms in the analysis (as an outgroup) brought some insight on their taxonomy. Trochochaeta multisetosa was originally described from the Øresund Strait, Sweden, by Örsted ([234], as Disoma multisetosum) and later occasionally reported from the North East Atlantic. Hartman [241] described Trochochaeta franciscana (as Disoma franciscanum) from San Francisco Bay, California, USA, but Pettibone [239] placed it into synonymy with T. multisetosa. In disagreement with Pettibone [239], Blake & Arnofsky ([242]: 68) stated that “the California specimens clearly represent a separate species due to differences in larval ciliary patterns.” In a revision of Trochochaeta, Radashevsky et al. [243] treated T. multisetosa and T. franciscana as two valid species. Molecular analysis performed in the present study, however, showed identity (p-distance = 0.00%) of the fragments of nuclear 18S and 28S rDNA sequenced in worms from Norway and Sweden and from Drake’s Bay, California, adjacent to San Francisco Bay. Consequently, in agreement with Pettibone [239], we tentatively suggest that T. franciscana is a junior synonym of T. multisetosa. As such, it may represent an introduced North Atlantic species in San Francisco Bay (where it was first collected in 1912, Hartman [241]) and nearby embayments, transported by vectors such as commercial oyster culture or shipping. This suggestion should be verified, however, by analyzing mitochondrial genes, such as COI and 28S rDNA, of individuals from the San Francisco Bay area, and by further analysis of additional populations, reported under the names of T. franciscana or T. multisetosa from a variety of habitats, from British Columbia to southern California, to further confirm if only one species of Trochochaeta is present.If confirmed, this may be a second case of the successful invasion of Trochochaeta into a remote region. Radashevsky et al. [243] recently suggested that Trochochaeta japonica Imajima, 1989 might have been introduced from the Asian Pacific to the estuary of Santos, São Paulo, Brazil, as larvae in ballast water of ocean-going vessels.