Limnodrilus hoffmeisteri, Claparede, 1862

Fend Svante Martinsson Christer Erséus, Yingkui Liu Steven V., 2017, Extensive cryptic diversity in the cosmopolitan sludge worm Limnodrilus hoffmeisteri (Clitellata, Naididae), Organisms Diversity & Evolution 17 (2), pp. 477-495 : 487-491

L. hoffmeisteri Claparède, 1862 , sensu stricto (species IX, Figs. 4 and 5)

L. hoffmeisteri Claparède, 1862, pp. 248–252 , plate I, figs. 1– 3, plate III, fig 12.

Several junior synonyms are likely to occur in the literature, but they are not yet recognized.

Neotype SMNH Type Coll. 8838, a DNA-barcoded, sexually mature specimen (CE22806) initially preserved in 80% ethanol: 21 anterior segments whole-mounted, compressed on slide; posterior part of the worm, was used for DNA extraction. COI barcode in GenBank, KY 369333; COI Haplotype 42 (Treebase accession 20383).

Type locality According to Claparède (1862), the original material was collected in Switzerland, B Dans le lit de la Seime près Villette , canton de Genève^ (the type locality). Neotype collected by Yingkui Liu in the same river (today called Seymaz), at 46.199° N, 6.194° E, on 24 Aug 2014.

Additional material examined All specimens fixed in ethanol and whole-mounted on slides, anterior segments only, except for CE12498 from China (for which a mid-body sample was taken for DNA extraction). From type locality: CE22807 (haplotype 46; COI barcode in GenBank KY 369624), CE22808 (Hap 45; barcode KY369334), CE22809 (Hap 46; barcode KY369335), and CE22811 (Hap 44; barcode KY369336). From Gothenburg, Sweden: CE3093 (Hap 42; barcode KY369520). From Wuhan, China: CE12493 (Hap 44; barcode KY369610) and CE12498 (Hap 48; barcode KY369611). For further information, see Supplementary Table S1 .

Other genetic information This species also contains COI Hap43 (CE4924; COI barcode in GenBank KY369547) and

Hap47 (CE1840; barcode KY369478). For further information, see Supplementary Table S1.

Geographical distribution Species is apparently widely distributed in the world; identified by us using combination of molecular and morphological data in material from Europe ( Switzerland, Germany, The Netherlands, and Sweden); one GenBank record from Italy (EU117546), Asia ( China, Japan, Malaysia, and Thailand), and North America ( Canada and USA).

Description of neotype Length about 20 mm (estimated from photo of complete worm taken before the DNA sample was removed), width 0.34 mm in segment VIII, to 0.36 mm in XI. Pharynx in segments II–III, chloragogen cells dense on gut beginning in V. Segments II–VIII with 5–8 (median 7) fully developed chaetae per bundle ( Figs. 4c and 5a), and often a partially developed replacement chaeta; post-clitellar segments mostly with 4–5 chaetae per bundle; dorsal and ventral bundles similar. All chaetae bifid, sigmoid, with nodulus at about the distal 1/3; teeth short and moderately divergent, approximately equal (4–5 μm long), or with upper tooth slightly longer; upper tooth usually slightly thinner than lower. Ventral chaetae absent in segment XI; no modified spermathecal chaetae in X. Chaetal length 100–132 μm in segments II–IX; 100–110 μm in middle segments, similar in dorsal and ventral bundles.

Clitellum weak, segments X–XII. Male pores in line with ventral chaetae, about level with dorsal chaetae in segment XI; pore opens into a simple penial bursa about 170 μm deep. Spermathecal pores in line with ventral chaetae, about midway between chaetae in segment X and anterior septum (9/10).

Sperm funnel conical, lips about 100 μm long, projecting into segment X from 10/11. Vas deferens difficult to follow, but at least 2600 μm long; diameter to 42 μm near atrium, somewhat thinner (26–36 μm) in middle portion, and narrower (26 μm) near sperm funnel. Vas deferens composed of a single layer of ciliated epithelium to 10 μm thick (thinner near sperm funnel), without obvious muscle layer. Atrium mostly in segment XII within gonadal sacs; narrowly fusiform, narrowing gradually to ejaculatory duct, and more abruptly to vas deferens; length 620 μm, diameter to 100 μm ental to prostate attachment, about 60 μm in ectal part ( Fig. 4a ). Atrium with a very thin (2 μm) muscle layer, and a thick epithelium; epithelial cells granular, irregular, with indistinct boundaries. Atrium joined near midpoint by a single, stalked prostate gland; prostate a fan-like cluster of irregular lobes; entire mass to 250 μm high; prostatic cells granular and indistinct. Ectal (ejaculatory) duct of atrium about 300 μm long by up to 30 μm wide, with a thin, non-ciliated epithelium; appearing slightly wrinkled and less evenly cylindrical than vas deferens.

Shaft of cuticular penis sheath nearly straight ( Figs. 4a, b , and 5k); wall thickness 2–3 μm. Sheath length 530 μm; diameter 48 μm at basal (proximal) end, tapering to 28 μm near middle, and only slightly narrower (26 μm) at distal end (below head); ratio of length to basal width 11. Distal end (B head^) of one penis sheath forms a plate 85 μm wide ( Figs. 4a and 5i, k), at most slightly wider on one side, one side bent upward; plate at a slight angle from shaft. Head of second penis sheath nearly circular, appearing more strongly angled from shaft ( Fig. 4b ), possibly from compression. Penis sheath surrounded by a close-fitting layer of codes: AT Austria, BE Belgium, CA Canada, CN China, DK Denmark, EE Estonia, FI Finland, DE Germany, HU Hungary, IN India, IT Italy, JP Japan, MY Malaysia, NO Norway, RO Romania, SE Sweden, CH Switzerland, TH Thailand, NL The Netherlands, UK United Kingdom, US United States of America epithelium (to 12 μm thick), continuous with lining of penial bursa and continuing as a thin layer within the sheath; entire structure surrounded by a thick layer (to 30 μm) of loosely arranged, transverse-spiral muscle fibers; near distal end, the muscles detach from sheath and join the body wall behind the male pore.

Spermathecal duct an irregular tube about 400 μm long, 80–120 μm thick, widest near ampulla. Duct composed of an irregular epithelium to 14 μm thick, surrounded by transverse-circular muscle fibers; ampulla irregular, filling much of segment. Spermatozeugmata 360–370 μm long, main part slightly tapered-cylindrical, about 50 μm wide in middle, distinctly widened at one end to 75–90 μm.

Description of other material Prostomium broadly conical or rounded. Segments II–VII or VIII with 4–8 (median 5–7) fully developed chaetae per bundle; usually 4–5 in mid-body (post-clitellar), posterior segments (in CE12498) with 1–2. Chaetae as above, teeth may be slightly larger (6–7 μm) in the worms from other sites ( Figs. 4e and 5b–d). Chaetal length 90–130 μm in segments II–IX; 80–100 μm in mid-body segments.

Pharynx in segments II–III, about equally developed dorsally and ventrally; chloragogen cells dense on gut, always beginning in segment V. Long, convoluted, lateral blood vessels visible in segments II–VII and in IX; in VIII, lateral blood vessels shorter and greatly dilated, modified as B hearts.^ Dorsal vessel ventrolaterally displaced in middle-posterior segments, beginning in segment X. In CE12498, one pair lateral blood vessels in posterior segments; network of capillary blood vessels not visible in epidermal layer.

Male and spermathecal pores as above; paired, inconspicuous transverse openings in line with ventral chaetae. Female pores inconspicuous, on 11/12. Clitellum weak, segments X– XII. Both testes and ovaries large, usually extending through X and XI, respectively.

Sperm funnel up to 200 μm high, on septum 10/11 or projecting into segment X. Vas deferens as above, very long and convoluted, up to about 3000 μm ( Fig. 4d, f ), diameter 24–36 μm in middle portion (Fig. 5f), but somewhat thinner (to 20 μm) near funnel, and thickest (to>40 μm) near atrium. Atrium as above; length 450–650 μm, maximum diameter 70–100 μm ental to prostate attachment, slightly thinner in ectal part ( Fig. 4d ). Atrium with a very thin (<5 μm), transverse muscle layer, and a thick (to 30 μm), granular epithelium. Prostate gland as above; entire mass to 200– 300 μm wide. Ectal (ejaculatory) duct of atrium variable, about 200–500 μm long by 20–30 μm wide, with a thin, non-ciliated epithelium surrounded by a thin muscle layer; indistinct in some specimens. Parts of the male duct, including the atrium, may extend into XII within the gonadal sacs ( Fig. 4f ).

Shaft of cuticular penis sheath slightly tapered from base to just below head; straight or slightly curved in slide-mounted specimens ( Fig. 4d, f ). Sheath length 420– 560 μm; diameter 44–54 μm at basal (proximal) end, gradually tapering to 23–28 μm at distal end (below head); ratio of length to basal width 8.4–11.2. Distal plate may be nearly circular (Fig. 5j) or wider and somewhat recurved on one side (Fig. 5h); diameter 60–110 μm. Plate orthogonal (Fig. 5g) or at a slight angle to shaft. Penial structure, including musculature, as described above.

Spermathecal duct variable; 200–300 μm long, usually narrowest near pore and expanding (to 120 μm thick) near ampulla ( Fig. 4d ). Duct composed of an outer, mostly transverse-circular muscle layer, and an irregular epithelium 15–40 μm thick. Spermathecal ampulla irregular, sacciform, to 390 μm wide (ampullae fill most of segment X), with thin wall (<10 μm). Spermatozeugmata 240–530 μm long, main body nearly cylindrical (30–55 μm wide), distinctly widened at one end, to 60–75 μm ( Figs. 4d , 5e).

Remarks This morphological description was intended to verify consistency with Claparède’ s (1862) original L. hoffmeisteri description. It is not an attempt to review or evaluate the vast morphological literature that has followed the original description. No original types are known to exist ( Reynolds and Wetzel 2015). When revisiting Claparède’ s original locality (Seymaz River, Canton Geneva) in August, 2014, four of the species now recognized as members of the L. hoffmeisteri complex (species II, VII, IX, and X), as well as L. claparedianus , were collected. In addition to this, Vivien et al. (2015), in a barcoding study of Swiss Naididae found species I and L. udekemianus in streams of the neighboring Canton of Vaud. Of these, species IX is the one that best conforms to Claparède’ s description of L. hoffmeisteri , and therefore, we selected a representative of this lineage from River Seymaz as a neotype of this taxon.

All specimens used in the description were sexually mature, with well-developed testes and ovaries, mature eggs in the egg sacs, sperm on the male funnels, and spermatozeugmata in the large spermathecal ampullae. Female funnels were not obvious in any specimen (which seems typical for the genus). Penis sheath form and proportions are perhaps the most consistently described characters in Limnodrilus literature; however, we note that the commonly reported length/width ratio varies greatly, depending on which part of the sheath is measured. Here, we use the basal (proximal) end of the sheath for width measurements, in keeping withrecent morphological literature. Proportions of penes in the new material, representing group IX specimens from three localities, are consistent with Claparède’ s illustration ( Claparède 1862, plate I, fig. 1). Claparède gave a B copulatory organ^ length/width ratio (possibly based on the terminal width near the distal part) of around 5–6, but measuring his drawing gives a penis sheath length/basal-width of about 11, as noted by Piguet (1913). Claparède’ s (possibly schematic) illustration shows a round (symmetrical) head, orthogonal to the shaft; in contrast, the head is asymmetrical in some of the present specimens, and the longer side may be somewhat reflexed. The apparent angle of the head may be related to either viewing aspect or compression in slide-mounting. However, penis sheaths are not strongly narrowed and curved distally in our material, and the distal ends do not widen abruptly to form a strongly angled hood as in the B typical^ form of L. hoffmeisteri shown in the recent guides ( Brinkhurst 1960, fig. 4G-H; Brinkhurst 1965, fig. 4a; Ohtaka 1985, fig. 5B; Ohtaka et al. 1990, fig. 3A–F; Hiltunen 1967, fig. 20; van Haaren and Soors 2013, fig. 230). Instead, they seem more similar to the B plate-topped^ or intermediate forms, e.g., fig. 5A in Ohtaka (1985), fig. 4 in Ohtaka et al.(1990).

Other characters related to morphology of reproductive organs are not emphasized in recent literature and have not been consistently described. The new material has a relatively elongate atrium, probably more similar to Claparède’ s illustration than to the atria of some other species (e.g., Limnodrilus profundicola [cf. figure 4 in ( Fend et al. 2016)]). Penial musculature is often prominent in the genus, with a characteristic spiral arrangement in some worms that have been attributed to L. hoffmeisteri ( Eisen 1885; Chen 1940; Moore 1905). This is clearly visible on the neotype and other material examined here, but Claparède’ s statement B composée de fibres longitudinales el de fibres circulaires^ does not clearly describe the same structure. Although the form of the spermathecal duct has been used as a taxonomic character within the genus in the past, the original description is rather vague. The neotype and associated specimens have a relatively elongate (tubular) spermathecal duct, compared with, e.g., L. profundicola ( Cui et al. 2015, fig. 7; Fend et al. 2016).

Although subsequent comparisons rely heavily on chaetal morphology, chaetae were barely mentioned by Claparède (1862): relatively slender, 6–8 anteriorly, 4–6 in mid-body, 2–3 posteriorly; according to the single illustration (plate 3, fig. 16), the upper tooth is slightly longer than the lower. Later works on species that have been attributed to L. hoffmeisteri have focused on length (particularly relative length) of the distal teeth: most commonly, teeth have been considered subequal, but with the lower tooth sometimes thicker (e.g., Southern 1909; Chen 1940; Brinkhurst 1965; Ohtaka 1985). Chaetal morphology seems rather consistent in the present material; although the upper tooth may be slightly longer than the lower, it is never distinctly longer or shorter; teeth are moderately divergent and not strongly curved.

Claparède’ s description included internal somatic characters that are difficult to observe in our alcohol-preserved material. There is no evidence of a highly vascularized body wall in posterior segments (as in L. udekemianus ) in the single specimen with some remaining posterior segments. The hearts in segment VIII only seem typical for the genus ( Chen 1940). Nephridia could not be seen clearly. In some guides (e.g., Timm 2009, van Haaren and Soors 2013), the dense, continuous layer of chloragogen tissue on the gut, beginning on segment V seems an important specific character of L. hoffmeisteri when separating this taxon from L. udekemianus , although Claparède (1862, p. 226) stated that the coating of chloragogen cells (cellules pigmentaires) begins on segment V in both species.

Figures

Fig. 4 Morphological characters of Limnodrilus hoffmeisteri sensu stricto (species IX), from slide-mounted specimens. a Neotype. Atrium, vas deferens and penis sheath. b Neotype.Second penis sheath. c Neotype. Chaetae in VII (some broken), a partly developed chaeta at bottom. d Specimen from China (CE12498). Reproductive organs. e–f