Stylodrilus tofaceus Rodriguez, Vuckovic & Kerovec 2020, n. sp.

Stylodrilus tofaceus Rodriguez, Vučković & Kerovec n. sp.

(Figures 4, 5)

Holotype. MNCN 16.03 /3105, one sagitally dissected individual, posteriorly incomplete, fully mature, stained in hematoxylin and mounted in Canada balsam, 6 November 2013.

Paratype. MNCN 16.03 /3106, one sagitally dissected, fully mature individual, and MNCN 16.03 /3107 one whole-mounted, with developed reproductive organs but unmated individual; both paratypes are complete, stained in hematoxylin and mounted in Canada balsam. From type locality, 6 November 2013.

Type locality. Roški slap, Croatia, Coordinates: 43°54’12.1”N, 15°58’31.3”E (43.90336 N, 15,975 348 E). Collector: Zlatko Mihaljević.

Etymology: species named from Latin “tôfus”, a term applied to several soft rocks including the calcareous tufa.

Further material investigated. 3 dissected individuals and 4 whole mounts, all fully mature, except for one whole-mount with developed reproductive organs but unmated, stained and mounted in Canada balsam, 6 November, 2013. 29 worms sampled in 6 November, 2013 and 15 worms sampled in 7 February, 2014, kept in alcohol 70%; all from the type locality but poorly fixed and unsuitable for histological studies (in P. Rodriguez collection).

Description (based on fully mature individuals, i.e. with sperm in spermatheca and/or atrium). Segment number 53–69 (on 4 complete individuals, including both paratypes). Diameter in segment X, 0.60–0.70 mm. Prostomium rounded, 138–217 μm long. Secondary annulations not visible in preclitellar segments, slightly marked from XI backwards (although perhaps due to poor fixation). Chaetae simple-pointed, two per bundle (Fig. 4 A–C). Anterior ventral chaetae 80–125 μm long, shortest in II, longer in posterior bundles (117–132 μm); chaetal width 4–6 μm (thinner in II, c. 3–4 μm); nodulus at about 0.4–0.5 from distal end; in the same segment, dorsal chaetae shorter than ventrals. In the rearmost section of the body (from about segment XXXV), chaetae thicker (5–7 μm) and distally more curved (Fig. 4C), with nodulus generally at one third from the tip of the chaeta. In the partially matured worms the same pattern is observed.

Clitellum from the line of chaetae in IX to the end of segment XIII, both dorsally and ventrally. One pair of male pores in X, behind ventral chaetae, opening closely to septum 10/11, and displaced medially from the line of ventral chaetae (Figs. 4E, 5A). One pair of spermathecal pores in IX, close to and medially of the ventral chaetae, on the transverse plane containing the chaetae (Figs. 4D, 5A). Female pores in the intersegment 11/12, in line with ventral chaetae.

Body wall formed by smooth epidermis (up to 17 μm high, to 27 μm at the clitellum), circular musculature (3–5 μm thick) and longitudinal musculature (16–20 μm thick dorsally and 27–50 µm ventrally). Pharynx with dorsal pharyngeal pad in II and III. Pharyngeal glands from IV to VI with dorsal, lateral and ventral lobes, and in VII only a small pair of ventral lobes. Chloragogen cells cover the gut from the posterior part of VI, backward (from V in one specimen). First nephridia at 6/7, and also observed at the ventral side of several postclitellar segments. Posterior lateral blood vessels with branches observed in only one specimen, not seen in the rest of material, probably due to bad fixation of posterior body section, which generally appears broken or beaded; thus, their presence and shape should be confirmed in new material.

Paired testes in IX and X, one pair of ovaries in XI. Sperm sac extending to VII anteriorly and to XIII poste- riorly, ovisac to XIV. One pair of semi-prosoporous atria in X, two vasa deferentia per atrium, with male funnels at 9/10 and 10/11, both functional with sperm (Fig. 5B). Vasa deferentia 15–18 μm in diameter, the anterior one entirely in X, the posterior one entering XI where it forms a loop before joining atrium. The vasa deferentia join the atrium separately but closely to each other at the basal third of the ampulla, run toward the apical part of the atrium, and open at the basal part of the ampulla into the atrial lumen. Atrium elongate (198–267 μm long), club-shaped, with ectal end gradually narrowed towards pore, its length about one half of the body diameter in X. Atrial ampulla 141–155 μm long, 64–84 μm in diameter; atrial muscle layer 5–10 μm thick, epithelium 5–8 μm high, ampulla covered by a layer of prostatic cells organized in small clusters, up to 45 μm high (Fig. 4F). Atrial duct not well separated from the ampulla, devoid of prostatic cells, about 110 μm long, 27–34 μm in diameter, and covered by a muscle layer (3–5 μm thick). Atrium opens in a simple epithelial invagination, with a conical penis (17–25 μm long, 27–34 μm diameter) (Figs. 4G, 5B).

One pair of spermathecae in IX (Fig. 5B). Sac-like spermathecal ampulla (254 μm long and 90 μm wide) with strongly vacuolated epithelium in its ental region. Spermathecal duct spindle-shaped, 113–149 μm long, maximum diameter 58–88 μm (narrowed at both ends of the duct to 23–25 µm), shorter than ampulla, with a narrow lumen and lining epithelium 28–40 μm high at the widest section, surrounded by a thin muscle layer (c. 2 μm) which becomes thicker distally (to 6 μm) (Figs. 4H, 5B). Female funnels at 11/12.

Remarks. The new species is morphologically close to other Stylodrilus species with simple-pointed chaetae and elongate atria (tubular, cylindrical or elongated) (see Table 3). Among these species, S. subcarpathicus (Hrabě, 1929) (not included in Table 3) is clearly separated from any other known Stylodrilus species by the very long, tubular atrium which extends to segment XII. The species S. glandulosus Giani & Martínez-Ansemil, 1984 and S. curvithecus Collado, Martínez-Ansemil & Giani, 1993 are also well separated from other congeneric species by the presence of long atria that pass to segment XI, and the atrial duct associated with a bulb surrounding a penial sac, i.e. an invagination of the body wall containing the penis. Stylodrilus species reported in the Table 3 are all of medium size (≤ 1 mm body diameter) and share the following characters: prostatic cells usually organized into clusters; junction of vasa deferentia to atrial ampulla basal, and opening to atrial lumen medially to apically; penis short and/or conical (absent only in S. sulci). The new species is mainly distinguished from other Stylodrilus species by the relatively small size of the atrium, similar in size to the atrium in S. coreyi Rodriguez et al., 2014 (see Table 3), the position of spermathecal pores in the transversal plane containing ventral chaetae and toward the ventral midline of the body, and the shape of the spermathecal duct, spindle-shaped, and formed by very high lining cells. Among Stylodrilus species known so far, spermathecal ducts are generally tubular, as in S. sulci (Fig. 4I) or tubular to barrel-shaped depending on the contraction of the muscular layer of the duct, as in S. lemani (Figs. 4J, K). Short, spindle-shaped spermathecal ducts with long lining cells were also described in S. mollis Timm, 1998, but this species has bifid chaetae and other differences in the structure of the atrium. Commonly, the spermathecal pores in Stylodrilus open behind the ventral chaetae and in line with them; the position of spermathecal pores in S. tofaceus n. sp. is similar to that in S. lemani (P. Rodriguez, personal observation).

Immatures in the same samples were ascribed to the species based on the form of the chaetae (simple pointed), position of pharyngeal glands (back to segment VII) and first nephridia (in 6/7), as well as the typical position of gonads when partially mature.