Enchytraeus albidus species complex
Diagnosis
Large Enchytraeus worms, > (7.5) 10 mm; high segment number (> 40); white to yellowish; ventral chaetae 3 or more in several bundles; esophageal appendages short, tube-like; vasa deferentia extending into segments posterior to clitellum (not always for E. polatdemiri); penial bulbs surrounded by several accessory glands. (E. polatdemiri is not further treated below.)
Enchytraeus albidus Henle, 1837 sensu stricto (Figs. 4–8)
E. albidus Henle, 1837: pp. 74–90, pl. VI, figs. 1–9; Bell 1958: pp. 2–11, figs. 1–10.
E. albidus partim; Nielsen and Christensen 1959: pp. 91–92, figs. 95–100.
E. constrictus Backlund, 1947: pp. 8–13, figs. 4–6, pl. II, figs. 7–10.
E. hortensis Goodrich, 1897: pp. 51–69, pl. V–VI, figs 1–15, 18–26, 28.
E. humicultor Vejdovský, 1879: p. 57, pl. V, figs. 1–11.
?E. pellucidus Friend, 1899: pp. 264–265.
E. pellucidus; Stirrup 1913: pp. 300–321, pl. XLVI, figs. 2, 4–5; pl. XLVII, figs. 10–13; pl. XLVIII, figs. 14–18; pl. XLIX, figs. 19–21.
?E. multiannulatus Altman, 1936: pp. 29–32, pl. XIII, figs. 108b, 109; pl. XIV, figs. 114, 117.
?E. multiannulatoides Altman, 1936: pp. 33–37, pl. XIII, figs. 105–108a.
Non E. sabulosus Southern, 1906: pp. 180–184, figs. 1–7 (strange spermathecae and chaetae; see “Remarks” below).
E. albidus “clade B”; Erséus and Gustafsson 2009.
E. albidus; Erséus et al. 2010.
Non E. albidus; Christensen and Glenner 2010: Table 1.
E. albidus “EA-SW, EA-I[1–2], EA-S[1–9]”; Arslan et al. 2018.
Non E. albidus “EA-DK”; Arslan et al. 2018.
Neotype
SMNH Type Collection 9122 (CE2169), mature specimen, whole-mounted on a slide, from lab culture, ECT Oekotoxikologie GmbH, Flörsheim, Germany, Dec 2006, leg. J. Römbke and A. Haller. COI barcode: GenBank MK266822. Figures 5a–d, 6, and 7a are illustrations of this specimen.
Other material examined morphologically
SMNH 172851 (CE2170), one specimen from the same lab culture as the neotype; SMNH 172854–172855 (CE2547 and CE2786), two specimens from Swedish seashores; and SMNH 172857–172861 (CE2866–2870), five specimens from a composting toilet in Sweden. 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 5-7 times longer than wide; vasa deferentia with uniform cell wall thickness; penial bulbs same size or larger than accessory glands; spermathecae sometimes with one or more diverticula.
External characters
Color white (Fig. 4). Length of first 16–40 segments > 4–12 mm (fixed, amputated specimens); first 12 segments (anterior end to clitellum) 2.6–3.3 mm long; width at clitellum 0.57–1.08 mm. Chaetae straight or slightly curved (Fig. 5c). Lateral bundles with 3–4(5) chaetae anterior to clitellum, 2–3 in XII, (2)3 chaetae in postclitellar segments. Ventral bundles with 3–5 chaetae anterior to clitellum, missing in XII, 2–4 chaetae in postclitellar segments. Chaetae longest in preclitellar ventral and lateral bundles (VIII–XI) measuring 80–130 μm long, about 5–8 μm wide. Clitellum extending over XII–½XIII (Fig. 5a). Head pore between prostomium and peristomium. Epidermis with transverse rows of gland cells. In some specimens, a deep transversal groove indenting the lateroventral body wall between segments IV and V; this is evidently the feature described by Backlund (1947: fig. 4).
Internal characters
Coelomocytes numerous, 10–20 μm long, round, oval or spindle-shaped, granulated, and with distinct nucleus. Paired pharyngeal glands in IV, V, and VI. First pair of glands small, third pair usually largest (Fig. 5a); second and third pairs converging dorsally, dorsal junction in first pair not always evident. Esophageal appendages (peptonephridia) extending from dorsal wall of esophagus in III. Dorsal vessel originating in XIII–XVII, usually XIV or XV. Nephridia in 6/7–9/10 and from 13/14 to 16/17 at least, 110–175 μm long, anteseptale consisting of funnel only, postseptale elongate ovoid, with posteroventral efferent duct (Fig. 5d). Brain longer than wide, posterior margin straight or slightly indented (Fig. 5b).
Male genitalia paired. Testes in XI, each surrounded by irregularly lobed mass representing different stages of spermatogenesis enclosed by peritoneal sac; testis sacs bulging forwards into X, sometimes into IX or even VIII (Fig. 5a). Sperm funnels in XI, 505–1085 μm long, 105–190 (240) μm wide at the widest point, making them about 5–7 times longer than wide, funnels tapering towards vasa deferentia. Vasa irregularly coiled in XII–XVIII or even XX, about 30–45 μm wide with 5–10 μm thick wall along ental and ectal portions, gradually widening towards mid portion, which makes up most of vas’ length and is about 35–55 μm wide with 10–15 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; male pores immediately beneath these lips (Fig. 6). Penial bulbs compact, round, 60–95 μm in diameter, sheathed with muscles and surrounded by numerous accessory glands that are smaller or about same size as bulbs (Figs. 6a–b). Ovaries in XII. About three to eight mature eggs present at a time.
Spermathecae in V, with ectal pores at lateral lines. Ectal duct of spermatheca abruptly widening into sac-like ampulla (Fig. 7a–d) laterally connected to esophagus; ampulla often bearing one or more sac-like diverticula. Sperm filling lumen of ampulla and diverticula, heads of spermatozoa embedded in walls of diverticula, forming aggregates. Spermathecae 175–370 μm long, 75–215 μm wide at widest part of ampulla. Ectal duct surrounded by gland cells forming compact mass 75–155 μm in diameter at its widest part; in some (possibly all; see “Remarks”) specimens, short, inner part of duct not covered by these cells. No obvious midventral subneural glands observed.
Specifics of neotype
Length of first 16 segments (fixed) > 4 mm; first 12 segments 3 mm long; width at clitellum 0.72 mm. Lateral bundles each with 3–4 chaetae anterior to clitellum, 3 chaetae in postclitellar segments. Ventral bundles each with 3–4 chaetae anterior to clitellum, 3 chaetae posteriorly. Chaetae up to 95 μm long, about 5 μm wide. Clitellum extending over XII–½XIII.
Coelomocytes numerous, 15 μm long. Pharyngeal glands with ventral and dorsal lobes, first pair with smallest ventral lobes, third pair with largest. Dorsal lobes converging dorsally in at least first and third pairs. Dorsal blood vessel originating in XIII (or XV?). Nephridia in 6/7–9/10 and 13/14–15/16, about 155 μm long.
Testes in XI, testis sacs extending forwards into IX. Sperm funnels 780 μm long, 105 μm wide at widest point, making them about 7 times longer than wide. Vasa irregularly coiled in XII–XIV, width from 30 μm (with 7 μm thick wall) in the proximity of penial bulbs to 35 μm (with 10 μm thick wall) in postclitellar loops. Penial bulbs compact, round, 70 μm in diameter, surrounded by accessory glands of about the same size or smaller than penial bulbs. Ovaries in XII. One mature egg present.
Spermathecae in V, narrow ectal duct abruptly widening into sac-like ampulla (Fig. 7a). In one spermatheca, ampulla seemed divided into two sacs, one visibly connected to esophagus. Spermathecae 180 μm long, 110 μm wide at widest part of ampulla. Ectal gland 100 μm in diameter at its widest part; innermost (short) part of duct clearly devoid of glands at least on one side (Fig. 7a). No obvious midventral subneural glands observed.
Remarks
In the DNA-based phylogeny of Enchytraeidae proposed by Erséus et al. (2010), the specimen CE521_1 was included to represent E. albidus, which is fortunate, as we have now been able to confirm that it belongs to E. albidus s. str. However, in a coeval molecular assessment of the family, Christensen and Glenner (2010) used a specimen of E. albidus (from Northern Zealand, Denmark), which according to its COI sequence (GenBank # GU453370) can now be identified as E. moebii (see below).
Our specimens (those studied morphologically) match the original description of E. albidus well in most characters, such as the number of chaetae, shape of nephridia and male genitalia, the latter with long sperm funnels. With regard to the coverage of glands along the spermathecal duct, we noted a short naked region near the base of the ampulla in some specimens (including the neotype), exactly as shown for various specimens depicted by Nielsen and Christensen (1959: figs. 95–97); however, we could not confirm this feature in all of our (fixed and whole-mounted) material.
E. albidus s. str. was originally described from potted soil (see the “Introduction” section). This is compatible with the habitat of our specimens, which were collected in terrestrial habitats, including lab cultures, as well as in seashores. It should be noted that all the other species of the E. albidus complex (except E. polatdemiri) are exclusively known from sites near the sea.
To clarify the taxonomic status of E. albidus s. str., we have decided to designate a neotype of this species, under the qualifying conditions stated by the International Code of Zoological Nomenclature, Article 75.3. (http://www.iczn.org/code). The lack of old type material and the uncertainty of the original locality were mentioned in the “Introduction” section above, but from Henle’s (1826) words, it appears that his material was from more than one German site, and more likely from a terrestrial habitat than the marine littoral. As we have access to sexually mature, DNA-barcoded specimens of E. albidus s. str. from a laboratory strain commonly used in Germany, we have assigned the name-bearing status to one of these specimens. This will stabilize the nomenclature of E. albidus, and permit the continued use of this name for the lineage most commonly used in applied studies (e.g., ecotoxicology). We present molecular evidence herein that the same strain as cultured for several years in Flörsheim (Germany) labs has also been used for scientific work in Aveiro (Portugal) and in other labs elsewhere in the world (J. Römbke, M. Amorim, pers. comm.). The cultures were part of an international ringtest during the development and validation of the Enchytraeid Reproduction Test (ERT; Römbke and Moser 2002). Unfortunately, it is not possible to trace the barcoded worms back to one specific geographic site or breeder.
E. albidus s. str. can be distinguished from the other species of the complex considered here by two main characters: the proportions of the sperm funnels and the morphology of the copulatory organs. The length/width ratio of the sperm funnels is about 5–7: 1 in E. albidus s. str., whereas in the other species this ratio is only about 1.5–4: 1. Furthermore, the penial bulbs in E. albidus s. str. are smaller than in E. moebii and generally smaller than in E. albellus sp. nov., and both specimens of E. cf. krumbachi that we examined (Table 3). The main glands (bulbs) in the penial apparati of E. albidus s. str. are in some cases larger but usually about the same size as the surrounding accessory glands, while the bulbs of E. moebii, E. cf. krumbachi, and E. albellus are always clearly larger than the surrounding glands. We also observed a higher variability in the shape of the spermathecae in E. albidus s. str. than in the other species, but this may be due to the fact that we studied a higher number of specimens of E. albidus.
Table 3 Comparison of morphological characters between the species included in this study. Measurements of E. polatdemiri are derived from text and figures in Arslan et al. (2018)
The earlier descriptions of the species already synonymized (i.e., E. constrictus Backlund, E. hortensis Goodrich, and E. humicultor Vejdovský) match our strict definition of E. albidus well in most characters. Backlund (1947) described his Enchytraeus constrictus as being distinguished from E. albidus by the deep intersegmental groove between segments IV and V. This character was observed in two out of nine of our specimens of E. albidus s. str., thus it may not be of much taxonomic value. Furthermore, the three synonymous taxa mentioned above were all described from terrestrial habitats, E. constrictus from a pile of manure in Sweden, E. hortensis from a garden in southern England, and E. humicultor from humid, ammonia-rich soils outside Prague in the Czech Republic. The other species we have recognized all have an exclusively marine littoral lifestyle; therefore, we agree with Nielsen and Christensen (1959) and previous authors in regarding E. constrictus, E. hortensis, and E. humicultor as junior synonyms of E. albidus s. str.
E. pellucidus Friend, 1899, also resembles E. albidus somewhat. This species was originally found in a pile of manure outside Manchester, England, and briefly described as having three–four chaetae per bundle, white color, and vasa deferentia extending back into XX or even XXIV. The chief distinction between this species and E. albidus s. str. is the lack of glands at the spermathecal openings. However, there is no illustration to support this, and the original specimens of E. pellucidus have been lost. The taxonomic status of E. pellucidus thus remains doubtful. In 1913, Stirrup re-described E pellucidus from a similar habitat (a heap of leaf-mold near Birmingham, England) and provided several informative illustrations, in particular the one showing a cross section of the genital fields of two specimens during copulation, where spermathecal glands (“sp. gl.”) are shown near the external opening in the sperm-receiving worm (Fig. 8). Stirrup stated that E. pellucidus indeed lacks the rosette of glands around the pore of the spermatheca, but also that this species does have simple glands along the ectal part of the spermathecal duct. After studying specimens of both E. pellucidus and E. albidus, he concluded that the two cannot be regarded as separate species.
In 1936, Altman described two Enchytraeus species: E. multiannulatus and E. multiannulatoides from Washington State, USA. Both are reminiscent of E. albidus and both were found in terrestrial habitats; E. multiannulatus from decaying organic matter and newspapers near the edge of a salt march, and E. multiannulatoides from a compost with manure. The two species are supposedly separated from E. albidus s. str. and each other by the number of chaetae per bundle (mostly 3 laterally and 5 ventrally in E. multiannulatus; mostly 4 laterally and 4–5 ventrally in E. multiannulatoides) and number of accessory glands (10 in E. multiannulatus; 18–20 in E. multiannulatoides) in the genital field, but future studies will be needed to prove their taxonomic status.
E. sabulosus Southern, 1906, described from gravel at the high-water mark in Dublin Bay, Ireland, has also been regarded a synonym of E. albidus (e.g., by Nielsen and Christensen 1959). However, as this species has never more than two–three chaetae per bundle, spermathecae with a thin duct “thickly covered with small glands all along its length”, sperm funnels three–four times as long as broad, and vasa deferentia extending backwards to XX but of unknown structure, we consider it separate from E. albidus.
Geographical distribution of genetically verified specimens
Germany, Greenland, Norway, and Sweden in the present study; also recognized from Ireland and Northern Spain (Arslan et al. 2018) (see “Habitat” below), and Canada (as COI barcodes among BOLD records). This species is represented in BOLD by BIN: AAN7506. Morphologically identified specimens (including species placed as synonyms above) indicate a wider range, but specimens from these localities have not been verified as belonging to E. albidus s. str. as defined here.
Habitat
Seashores (above or below the high-water mark), salt marshes, and terrestrial; typically supralittoral, in decomposing seaweed and algae, or in decomposing organic material on land. Occasionally in freshwater. Worms can be kept for years in laboratory cultures.
The records from Ireland and Northern Spain in Arslan et al. (2018) were from lab cultures maintained by Dr. Rüdiger M. Schmelz since the original collection in the field (1995, Bull Island, Dublin, and 2011, Las Amorosas, A Coruña, respectively). According to Schmelz (pers. comm.), the Irish site was upper littoral, a sandy soil flooded at highest tide, and the Amorosas site was slightly more terrestrial, i.e., turf of a thrift species (Armeria pubigera) on solid granite, but within the reach of sea spray. This shows that wild strains of this species may live in a lab regardless of their original habitat, i.e., with or without access to salt from the sea.
Enchytraeus moebii (Michaelsen, 1885) (Figs. 9–10)
Archenchytraeus möbii Michaelsen, 1885: pp. 237–239.
Enchytraeus möbii; Michaelsen 1886: pp. 1–52, pl. I, figs. 1–16, pl. II, figs. 1–7, pl. III, figs. 1–10.
E. albidus partim; Nielsen and Christensen 1959: pp. 91–92, figs. 95–100.
E. albidus “clade A”; Erséus and Gustafsson 2009.
E. albidus; Christensen and Glenner 2010: table 1.
E. albidus “EA-DK”; Arslan et al. 2018.
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.
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.
Enchytraeus albellus Klinth, Erséus and Rota, sp. nov. (Fig. 11
)
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.
Enchytraeus cf. krumbachi (Čejka, 1913) (Fig. 12)
?Litorea krumbachi Čejka, 1913: pp. 145–151, figs. 1–10.
?E. albidus partim; Lasserre and Erséus 1976: pp. 452–453.
E. albidus “clade C”; Erséus and Gustafsson 2009.
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.
Enchytraeus sp. 1
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.