Enchytraeus albidus Henle, 1837

Erséus, Christer, Klinth, Mårten J., Rota, Emilia, De Wit, Pierre, Gustafsson, Daniel R. & Martinsson, Svante, 2019, The popular model annelid Enchytraeus albidus is only one species in a complex of seashore white worms (Clitellata, Enchytraeidae), Organisms Diversity & Evolution (New York, N. Y.) 19 (2), pp. 105-133 : 117-122

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 B Remarks^ below).

E. albidus B clade B^; Erséus and Gustafsson 2009.

E. albidus ; Erséus et al. 2010.

Non E. albidus ; Christensen and Glenner 2010: Table 1.

E. albidus B EA-SW , EA-I[1–2], EA-S[1–9]^; Arslan et al. 2018.

Non E. albidus B 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

numbers indicate different alleles from the same specimen). c ITS2. In the trees, posterior probability support values are given at the branches; scale bars indicate expected number of substitutions per site

( 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 IVand 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 B 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 B 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 B 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 .

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 redescribed E pellucidus from a similar habitat (a heap of leafmold 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,

N/O not observed (lack of mature specimens)

where spermathecal glands (B 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. m u l t i a n n u l a t u s an d E. multi an nul ato ide s fr om 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 B 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 B 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.

Figures

Fig. 4 Enchytraeus albidus s. str., live specimen, from the same locality as CE20518 (see Table 1). Photo: C. Erséus

Fig. 5 Enchytraeus albidus s. str. a Internal morphology of segments I– VI and IX–XIV viewed from dorsal side. b Brain. c Bundle of chaetae. d Nephridium. ag accessory glands, as anteseptale, br brain, cl clitellum, nd nephridial efferent duct, ov ovary, pb penial bulb, pg pharyngeal glands (only ventral lobes illustrated), ph pharyngeal pad, ps postseptale, s spermatheca, sf sperm funnel, t testis, vd vas deferens. Scale bars 100 μm

Fig. 6 Enchytraeus albidus s. str. a Male apparatus viewed dorsally. b Schematic illustration of the male apparatus in cross section. ag accessory glands, pb penial bulb, vd vas deferens. Scale bar 100 μm

Fig. 7 Enchytraeus albidus s. str. a–d Spermathecae. ed ectal duct, eg ectal gland, sa spermathecal ampulla, sd spermathecal diverticulum, sm sperm. Scale bars 100 μm

Fig. 8 Enchytraeus pellucidus Friend, 1899, sensu Stirrup (1913), section through two specimens during copulation (reproduced from Stirrup 1913, pl. XLIX, fig. 19)