The popular model annelid Enchytraeus albidus is only one species in a complex of seashore white worms (Clitellata, Enchytraeidae)

Material examined

SMNH 172878–172880 (CE965, CE972, and CE973), SMNH 172883 (CE2789), and SNHM 172885 (CE2966), 5 specimens from Swedish seashores; SMNH 172876 (CE1686), 1 specimen from algal compost in Galicia, Spain. All specimens sexually mature and COI barcoded. For more details, including GenBank accession numbers for genetic data, see Table 1.

Diagnosis

Several chaetal bundles with more than three chaetae; sperm funnels 1.5–3.5 times longer than wide; vasa deferentia with uniform cell wall thickness; penial bulbs larger than accessory glands; spermathecae without diverticula.

External characters

Color white. Length of first 17–35 segments, > 3–9 mm (fixed, amputated specimens); first 12 segments (anterior end to clitellum) 2.3–3.8 mm long; width at clitellum 0.43–0.89 mm. Chaetae straight or slightly curved. Lateral bundles with 3–4 chaetae anterior to clitellum, 2 in XII, 2–3 chaetae in postclitellar segments. Ventral bundles with 3–4(5) chaetae anterior to clitellum, missing in XII, 2–3 chaetae in postclitellar segments. Chaetae longest in ventral bundles anterior and posterior to XII, measuring 60–120 by 5–8 μm. Clitellum extending over XII–½XIII or –¾XIII. Head pore not observed. Epidermis with transverse rows of gland cells.

Internal characters

Coelomocytes numerous, 10–15 μm long, round, oval, or spindle-shaped, granulated and with distinct nucleus. Paired pharyngeal glands present in IV, V, and VI. All pairs with secondary lobes, first and second pairs possibly with narrow dorsal connection, third pair not connected. Esophageal appendages (peptonephridia) extending from dorsal wall of esophagus in III. Dorsal vessel seemingly originating in XIV or XV. Nephridia in 6/7–9/10 and from 13/14 to 21/22 at least, about 80 μm long, anteseptale consisting of funnel only, postseptale elongate ovoid, with efferent duct originating posteroventrally. Brain posterior margin straight or slightly indented.

Male genitalia paired. Testes in XI, paired, each enclosed in a sac and extending forwards into X. Sperm funnels in XI, 295–420 μm long, 120–265 μm wide at the widest point, making them about 1.5–3.5 times longer than wide, funnels tapering towards vasa deferentia. Vasa irregularly coiled in XII–XVI, of about uniform width (20–25 μm) throughout, with 2.5–5 μm thick wall. Vasa ciliated, without conspicuous musculature. Vasa seemingly not penetrating penial bulbs. Ventral surface of XII with invaginations creating two recesses with overhanging lips. Penial bulbs compact, round, 110–180 μm in diameter, sheathed with muscles, and surrounded by accessory glands much smaller in size (Fig. 9a). Ovaries in XII. About one to five mature eggs present at a time.

Spermathecae in V. Ectal pore at lateral line or just above. Ectal duct short to moderately long, covered with gland cells and abruptly widening into sac-like ampulla (Fig. 9b–c). Ampulla usually rounded, ental connection with esophagus uncertain, no observed diverticulum. Sperm in lumen of ampulla and sometimes in ectal duct; heads of spermatozoa embedded in wall of ental part of ampulla, forming aggregates. Spermathecae 205–225 μm long, 80–90 μm wide at widest part of ampulla. Gland cells surrounding ectal duct, forming compact mass 80–125 μm in diameter at its widest part; glands seemingly extending along entire duct (but see Fig. 10, and “Remarks”). No obvious midventral subneural glands observed.

Remarks

Our specimens agree for the most part with Michaelsen’s extended description of E. moebii (1886), but they have on average fewer chaetae per bundle and sometimes shorter sperm funnels. Our material is identified as this species primarily based on the combination of penial bulbs being larger than the accessory glands, and spermathecae having duct and ampulla of equal length but without diverticula. Obviously using sectioned material, Michaelsen (1886) illustrated a short inner part of the spermathecal duct as being devoid of gland cells (Fig. 10). This could not be discerned in our slide-mounted specimens, but it may be a general feature of this taxon.

E. moebii was synonymized with E. albidus by Nielsen and Christensen (1959), but the molecular data in this study support considering the two as separate species. Furthermore, unlike E. moebii, E. albidus s. str. has penial bulbs of about the same size as the accessory glands, sperm funnels quite elongate in relation to their width, and spermathecae with diverticula, making it possible to distinguish these two species morphologically.

E. moebii is morphologically most similar to E. albellus sp. nov. (described below), and these two species do not seem to be distinguishable with regard to chaetal size and number, or sperm funnel proportions (Table 3). Both species have penial bulbs that are much larger than the surrounding accessory glands (compare Figs. 9a, 10, and 11d). However, the spermathecae of E. moebii lack diverticula, whereas those of E. albellus usually have at least one dorsal diverticulum. Furthermore, the vasa deferentia of E. moebii have rather uniform width and wall thickness, whereas the middle portion of the E. albellus vasa is wider and has thicker walls than the ental and ectal portions. The dimensional contrasts between the different sections of the vasa deferentia are even more prominent in our specimens of E. cf. krumbachi (described below), which also makes the latter species distinguishable from E. moebii.

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E. moebii was originally described from decomposing seaweed in the Kiel Bay, Baltic Sea. Our specimens were partly from the Baltic Sea (sites around the island of Öland), but we also found the same lineage along a long stretch of the North-East Atlantic coast. We have decided not to designate any neotype for this species as we do not have material from the type locality.

Geographical distribution of genetically verified specimens

Norway (including a BOLD record from Oslofjorden, NOENC236-15), Sweden, and Spain in the present study; also recognized from Denmark (GenBank GU453370; see Christensen and Glenner 2010). This species is represented in BOLD by BIN: AAM5959. The type locality is on the Baltic coast of Germany.

Habitat

Seashores and salt marshes, but also in the intertidal zone; typically in decomposing seaweed and algae.

Holotype

SMNH Type Collection 9123 (CE6100), mature specimen, whole-mounted on a slide, from Färlevfjorden (inner end of Gullmarfjorden), west coast of Sweden, 27 May 2009, leg. C. Erséus, A. Ansebo, and M. Johansson. COI barcode: GenBank MK266873; for more details, including GenBank accession numbers for additional genetic data, see Table 1. Figures 11a–e are illustrations of this specimen.

Paratypes

All from type locality, mature, and whole-mounted on slides. SMNH Type Collection 9124–9126 (CE6101–6103); for more details, including GenBank or BOLD accession numbers for genetic data, see Table 1.

Other material studied

SMNH 172888–172889 (SM144 and SM189) two whole-mounted, mature specimens from a sandy, stony beach, Qeqertarsuaq town, Greenland; for more details, including GenBank numbers for genetic data, see Table 1.

Etymology

Latin albellus, diminutive of albus, i.e., whitish.

Diagnosis

Several chaetal bundles with more than three chaetae; sperm funnels 2–4 times longer than wide; vasa deferentia tripartite: ental and ectal sections thin-walled, middle section thick-walled, all parts ciliated; penial bulbs larger than accessory glands; spermathecae sometimes with one diverticulum.

External characters

Color white. Length of first 21–54 segments >3–10 mm (fixed, amputated specimens); first 12 segments (anterior end to clitellum) 1.6–2.7 mm long; width at clitellum 0.51–0.72 mm. Chaetae straight or slightly curved. Lateral bundles with 3–4 chaetae anterior to clitellum, 0–2 in XII, 2–3(4) chaetae in postclitellar segments. Ventral bundles with 3–5 chaetae anterior to clitellum, missing in XII, 2–3(4) chaetae in postclitellar segments. Chaetae longest in ventral preclitellar bundles (IV–XI) and some lateral bundles close to XII, measuring 65–115 by 5–8 μm. Clitellum extending over XII–½XIII or –¾XIII. Head pore between prostomium and peristomium. Epidermis with transverse rows of gland cells.

Internal characters

Coelomocytes numerous, 10–25 μm long, round, oval, or spindle-shaped, granulated and with distinct nucleus. Paired pharyngeal glands present in IV, V, and VI. All pairs with secondary lobes, dorsal connections between the pairs uncertain. Esophageal appendages (peptonephridia) extending from dorsal wall of esophagus in III. Dorsal vessel originating in XIV or XV. Nephridia in 6/7–9/10 and from 13/14 to 14/15 at least, about 75–80 μm long, anteseptale with funnel only, postseptale oval tapering into posteroventral efferent duct, in one specimen nephridia observed also from 30/31 to 49/50. Brain truncate posteriorly.

Male genitalia paired. Testes in XI, each surrounded by masses of cells at different stages of spermatogenesis within peritoneal sac; testis sacs bulging into IX. Sperm funnels in XI, 400–530 μm long, 135–235 μm widest, making them about 2–4 times longer than wide, funnels tapering towards vasa deferentia (Fig. 11a). Vasa irregularly coiled in XII–XXVIII, tripartite, ental and ectal parts thinner and thin-walled, 25–35(45) μm wide with 2.5–5 μm thick wall, middle part wider (30–45(65) μm) with thicker wall (10–20 μm) (Figs. 11b–c). All parts ciliated, without conspicuous musculature. No abrupt transition between thin- and thick-walled parts and the different parts seem indistinguishable in some specimens, possibly becoming clearer with maturation. Vasa seemingly not penetrating penial bulbs. Ventral surface of XII with invaginations creating two recesses with overhanging lips. Penial bulbs compact, round, 70–120 μm in diameter, surrounded by accessory glands much smaller in size (Fig. 11d). Ovaries in XII. About one to four mature eggs present at a time.

Spermathecae in V. Pore at lateral line. Ectal duct short, abruptly widening into sac-like ampulla (Fig. 11e) entally connected with lateral side of esophagus. Ampulla usually with irregular outline, sometimes with one, usually dorsal diverticulum. Sperm in lumen of ampulla, heads of spermatozoa embedded in wall of ental part of ampulla, forming aggregates. Spermathecae 125–335 μm long, (40)100–215 μm wide at widest part of ampulla. Gland cells surrounding ectal duct, forming compact mass 65–120 μm in diameter at its widest part; in some (possibly all) specimens, a short, ental part of duct not covered by these cells. No obvious midventral subneural glands observed.

Remarks

Despite a thorough literature search, we have been unable to locate any previous reference to, or description of, the E. albellus morphotype. E. albellus is genetically well separated from our specimens of E. albidus s. str. in both the COI (Fig. 3a) and ITS2 (Fig. 3c) regions. In addition, there are several morphological characters that distinguish E. albellus from all other species in the E. albidus species complex.

As noted above, E. albellus is morphologically similar to E. albidus s. str. and to our E. cf. krumbachi in having long, tripartite, and partly very wide and thick-walled vasa deferentia, which occupy several segments beyond the clitellum; E. moebii also has long vasa, but in this species the vasa are uniformly narrow.

E. albellus differs from E. albidus s. str. in the following characters: (1) the separation of the vasa into thick- and thin-walled sections is not as clear-cut in E. albidus as in E. albellus; (2) the penial bulbs are not much larger than the accessory glands in E. albidus, but the penial bulbs are distinctly larger than the accessory glands in E. albellus (compare Figs. 6 and 11d); and (3) the sperm funnels are 5–7 times longer than wide in E. albidus, compared to 2–4 times for E. albellus.

E. albellus can be separated from E. cf. krumbachi in having a dorsal diverticulum on the spermatheca, cilia in all parts of the vasa, and more than two chaetae per bundle in postclitellar segments (Table 3).

Geographical distribution

Sweden, Norway, and Greenland. This species is represented in BOLD by BINs: AAT8961 and ACV8067.

Habitat

In seashores, both above and below the high-water line, and with a clear tendency to go lower into the intertidal zone than E. albidus and E. moebii; occurs in decaying organic material on beaches, but is common also in rather clean intertidal sand and gravel.

Material examined

SMNH 172891-172892 (CE1684 and CE1689), two sexually mature and COI-barcoded specimens from algal compost in Galicia, Spain. For more details, including GenBank accession numbers for genetic data, see Table 1.

Diagnosis

A few chaetal bundles with more than three chaetae; sperm funnels at least 1.5–2 times longer than wide; vasa deferentia tripartite, ental and ectal sections thin-walled, middle section thick-walled and lacking ciliation; penial bulbs larger than accessory glands; spermathecae without diverticula.

External characters

Color white. Length of first 16–19 segments > 2 mm (fixed, amputated specimens); first 12 segments (anterior end to clitellum) 2.9–3.8 mm long; width at clitellum 0.40–0.62 mm. Chaetae straight or slightly curved. Lateral bundles with three chaetae anterior to clitellum, two chaetae in XII and postclitellar segments. Ventral bundles with three–four chaetae anterior to clitellum, missing in XII, two chaetae in postclitellar segments. Chaetae longest in preclitellar ventral bundles (VIII–XI), 55–75 μm long, about 5 μm wide. Clitellum extending over XII–¾XIII. Head pore not observed. Epidermis with transverse rows of gland cells.

Internal characters

Coelomocytes numerous, 10–15 μm long, round, oval, or spindle-shaped, granulated and with distinct nucleus. Paired pharyngeal glands present in IV, V, and VI. All pairs seemingly connected dorsally and possessing secondary lobes. Esophageal appendages (peptonephridia) extending from dorsal wall of esophagus in III. Origin of dorsal vessel not observed. Nephridia observed in 6/7–9/10, about 110 μm long, with oval postseptale tapering into posteroventral efferent duct. Brain truncate posteriorly.

Male genitalia paired. Testes in XI, paired, each enclosed in sac containing different stages of spermatogenesis; sacs extending forwards into IX. Sperm funnels at least 215–305 μm long, 135–160 μm wide at the widest point, making them at least 1.5–2 times longer than wide, tapering towards vasa deferentia. Vasa irregularly coiled in XII–XVIII, tripartite, ental and ectal portions thinner, 15–30 μm wide with 2.5–5 μm thick wall, widening to thicker mid portion, 50–55 μm wide with 15 μm thick wall (Fig. 12a). The transition between portions gradual, but with an abrupt change in the thickness of the duct wall; ciliation only in parts with thin wall. All parts lacking conspicuous musculature. Ventral surface of XII with invaginations creating two recesses with overhanging lips. Penial bulbs compact, round, 85–90 μm in diameter, not pierced by vasa, surrounded by accessory glands of smaller size (Fig. 12b). Ovaries in XII. About one to five mature eggs present at a time.

Spermathecae in V. Ectal pore at lateral line. Ectal duct thin, entally opening into a small, rounded chamber, distinctly set off from round ampulla; ampulla of similar length as entire duct (Fig. 12c). Ampulla round, without diverticula, connected to lateral side of esophagus. Sperm in lumen of ampulla. Spermathecae 155–180 μm long, 95–125 μm wide at widest part of ampulla. Gland cells surrounding ectal duct, forming compact mass 60–65 μm in diameter at its widest part; small chamber devoid of gland cells. No obvious midventral subneural glands observed.

Remarks

Our association of this species with the Mediterranean L. krumbachi Čejka, 1913, from a beach in Rovinj (Croatia), is largely based on the tripartition of the vasa deferentia. The three parts have different widths and wall thicknesses, and cilia are absent in the middle, thickened, tracts, conforming with Čejka’s observation. Similarly, Lasserre and Erséus (1976, plate 1C) showed a cross section of the thick-walled, but unciliated, part of a vas deferens in their Bermudian form of “E. albidus” (see also below). We observed that the vasa deferentia change in width over their length also in E. albidus s. str. and E. albellus sp. nov. In E. albidus s. str., they gradually (and slightly) widen from the ental to the middle part and then taper ectally, without any clear differentiation in wall thickness between the parts. In E. albellus, the difference in width between the ental, middle, and ectal parts is not as distinct as in our E. cf. krumbachi, but the middle part clearly has a thicker duct wall than the ental and ectal parts. However, E. albellus has cilia all along the vas’ walls, and spermathecae with diverticula, two features distinguishing it from E. cf. krumbachi.

Nevertheless, there are some morphological discrepancies between our Galician material and Čejka’s species. First, our form has fewer postclitellar chaetae per bundle; Čejka’s (1913) form had three chaetae in lateral, four chaetae in ventral bundles. Second, in a horizontal section of segment V (ibid., fig. 9), Čejka did not observe any separate chambers at the inner end of the spermathecal ducts (cf. our Fig. 12), although, in the same figure, the spermathecal ampullae were shown as barely wider than the glandular ducts, and his specimen may have been in a stage, where such chambers were still undeveloped.

The combination of the distinctly separated parts of the vasa deferentia, latitude of collection, and the adiverticulate spermathecae prompted us to denote our species as E. cf. krumbachi. And besides, based on the morphological variation within the E. albidus complex revealed here, and the more stringent definition of E. albidus proposed above, the proposed synonymization of E. krumbachi with E. albidus (see Lasserre and Erséus 1976) does not seem to be justified. However, it would be premature to formally attach the name E. krumbachi (without “cf.”) to our Galician specimens, considering that (1) we have no access to topotypes of the Adriatic E. krumbachi, (2) the number of postclitellar chaetae per bundle are fewer in our specimens than in the original description, and (3) additional forms within the E. albidus complex with the distinctly tripartite vasa deferentia are known from seashores of the North Atlantic/Mediterranean area; however, these forms have not been described in sufficient detail to establish whether or not our Galician specimens are conspecific with any of these proposed species.

There are at least three other literature reports of white worms with bi- or tripartite vasa deferentia of the kind described here. In 1874, Verrill described the species H. littoralis from the coast of Massachusetts (USA), in the Northwestern Atlantic. His brief morphological description (in Verrill and Smith 1874: 329–330) was later amended by Smith (1895), who transferred the species to Enchytraeus and described a long vas deferens with expanding thickness going from quite thin to one fifth the diameter of the entire worm in that region. Due to the pronounced thickening of the vasa deferentia, we do not consider E. littoralis as a junior synonym of E. albidus, as suggested by Michaelsen (1900). Furthermore, we had no access to topotypical material of E. littoralis, and thus, we cannot assess whether these two species are similar in other characters.

As noted above, Lasserre and Erséus (1976) referred to worms found on subtropical Bermuda, i.e., about 1200 km SSE of the type locality of E. littoralis, as E. albidus. The Bermudian worms also had massive outer parts of the vasa deferentia (op.cit.: pl. 1C): the latter were 65–70 μm wide, at least one fifth of the total body diameter, and with 20–25 μm thick walls. These measurements match rather well with the ones we observed for both E. cf. krumbachi (up to 55 μm wide, wall up to ca. 15 μm thick) and E. albellus (up to 65 μm wide, wall up to 20 μm thick). Lasserre and Erséus did not provide a complete morphological description, and we have neither new morphological nor any genetic data for the Bermudian form. Nevertheless, with regard to the similarities in the vasa deferentia, we cannot rule out the possibility that this form was conspecific to one of the two aforementioned taxa. If so, E. cf. krumbachi is more likely to be conspecific with the Bermudian specimens, as its latitudinal distribution is more similar. By contrast, the distribution of E. albellus extends into the High Arctic, which may indicate that this species is better adapted to a colder environment.

The third report is the description of Enchytraeus mediterraneus Michaelsen, 1926 from the coast of Tunisia. In this species, the width of the vas’ thick portion measured up to 85 μm. However, spermathecal ectal glands are absent in E. mediterraneus but present in our Galician worms (see more under “Remarks” to Enchytraeus sp. 1); we therefore do not consider our Galician specimens to be conspecific with E. mediterraneus.

The Galician E. cf. krumbachi was recovered as the sister species of Enchytraeus sp. 1 (see Fig. 2) from the Aegean Sea in the present molecular study, suggesting that the two represent a southern lineage (as opposed to the northern group of E. albidus, E. albellus, and E moebii).

Geographical distribution and habitat

Enchytraeus cf. krumbachi has been genetically identified from Spain only (present material). Čejka’s (1913) original material of E. krumbachi was from a seashore in Croatia, while our Spanish worms come from an algal compost.

Material examined

SMNH 172893-172894 (CE4859-CE4860), two half mature specimens from Skopelos Island, Aegean Sea, Greece. For information on voucher collection locality and GenBank accession numbers, see Table 1. Skopelos, Perivouli beach, NE of Glossa, 39° 11.84′ N, 023° 36.90′ E, in high intertidal sand, leg. C. Erséus, 21 Aug 2008.

External characters

Color white. Length of first 28–45 segments > 4–8.4 mm (fixed, amputated specimens); first 12 segments (anterior end to clitellum) 1.6–2.2 mm long; width at clitellum 0.46–0.77 mm. Chaetae straight or slightly curved entally. Lateral and ventral bundles with three–four chaetae anterior to clitellum, three chaetae in postclitellar segments. Chaetae longest in preclitellar ventral bundles (VIII–XI) measuring 125–135 by about 10 μm. Clitellum not developed. Head pore between prostomium and peristomium. Epidermis with transverse rows of gland cells.

Internal characters

Coelomocytes about 15 μm long, round, oval or spindle-shaped, granulated and with distinct nucleus. Paired pharyngeal glands present in IV, V, and VI. All pairs converging dorsally, first pair small, second pair largest. Esophageal appendages (peptonephridia) extending from dorsal wall of esophagus in III. Dorsal vessel seemingly originating in XV. Nephridia difficult to discern but pores of nephridial efferent ducts possibly observed in 5/6–7/8, and 8/9 and in some postclitellar segments, shape uncertain. Brain longer than wide, posterior margin straight.

Male genitalia paired. Developing testes in XI, penial bulbs and ovaries in XII. Rudimentary spermathecae in V.

Remarks

Although this Aegean Sea species is yet unidentified, and no details about its genital characters are known, the DNA-based phylogeny (Fig. 2) suggests that it is likely to be part of the E. albidus complex, and more closely related to E. cf. krumbachi than to the other species included here. Geographically, the record of E. sp. 1 is closest to the type locality of E. krumbachi in the Adriatic Sea (Čejka 1913).

According to the original description (op.cit.), E. krumbachi has lateral bundles with three chaetae and ventral bundles with four chaetae. Both our specimens of E. sp. 1 have three chaetae in all postclitellar bundles, both ventrally and laterally, but these differences may be due to the fact that our specimens are not mature.

Interestingly, the size of the chaetae in E. sp. 1 is the largest recorded in this study and recalls that of E. mediterraneus, described by Michaelsen (1926) from Posidonia detritus in Djerba (Southern Tunisia). E. mediterraneus is a very large worm (26 mm long, with 90 segments), with 3–4 chaetae in all bundles (each chaeta measuring 150 by 10 μm at midbody), pharyngeal glands dorsally fused and with large ventral lobes, dorsal vessel from XVIII, sperm funnels three times longer than wide, vas deferens reaching XXI and with diameter measuring 40–50 μm entally, 85 μm along the mid portion and 27 μm near the male apparatus, spermathecal ampulla onion-shaped with some small indistinct ectal swellings, ectal duct as long as ampulla but much thinner, and completely devoid of glands. As our specimens are immature, most of these characters cannot be compared to the description of E. mediterraneus. We therefore prefer to keep this Aegean material unidentified until fresh material of E. mediterraneus can be examined and barcoded.

Distribution and habitat

Known only from the high intertidal of a beach on the island of Skopelos, Greece. The sand at this site appeared poor in decaying organic material.