Holotype : Ma. 5, slide No. 3055. Type locality: (Loc. 3.) Italy, Punta Ala Grosseto, Castiglione della Pescaia, decaying seagrass detritus, 42°46'00.0"N, 10°51'31.0"E, Leg. András Dózsa-Farkas and Kinga Dózsa-Farkas, 24 Sep 2020.

Paratypes : in total, four specimens: P.146.1 slide No. 3022, P.146.2 slide No. 3056, P.146.3 slide No. 3077, P.146.4 slide No. 3192. Same data as for holotype, 24 Sep 2020 and 26 Nov 2020.

Four specimens for DNA analysis, five specimens only in vivo.

(1) Small size (body length 2–2.5 mm, 130–185 µm wide at clitellum, in vivo), segment number 19–30; (2) chaetae straight with ental hook, two chaetae in all bundles; (3) clitellum saddle-shaped; (4) first and second pharyngeal glands united dorsally, in V with ventral lobes; the third pair free dorsally with elongated ventral lobes; (5) dorsal vessel from clitellar region, blood colorless, anterior blood vessel bifurcation anteriorly behind the pharynx; (6) three pairs of preclitellar nephridia; (7) coelomocytes disc- or lemon-shaped with granules, 13–20 μm; (8) sperm funnels small, cylindrical, 100–140 μm long in vivo, 1.5–2.5 times longer than wide in vivo, collar high and narrower than funnel body; (9) spermatozoa 38–43 µm long, heads 15–22 µm in vivo; (10) male copulatory organs small and compact; (11) small subneural glands in XIII–XIV; (12) ectal duct of spermatheca surrounded along the length by glands and one larger, 15–27 µm long, sessile gland at orifice. Ampulla oval, 24–40 µm wide and 40–55 µm long in vivo.

Small species (Fig. 1A), holotype 1.9 mm long, 80 µm wide at VIII and 107 µm at clitellum (fixed), segment number 21. Body length of paratypes 2.0–2.5 mm, width 120–180 µm at VIII and 130–185 µm at clitellum, in vivo, length of fixed specimens 1.5–2.7 mm, width 80–165 µm at VIII and 105–165 at clitellum, segment number 19–30. Chaetae straight with ental hook. Chaetal formula: 2 - 2: 2 - 2 (in one specimen from Castiglione della Pescaia, three chaetae were in one ventral bundle). The chaetae equal in size within the bundles; in the ventral bundles a little longer than in the lateral ones. 15–20 μm in preclitellar segments and 19–20 μm at the posterior end of the body. Clitellum saddle-shaped in XII-1/2 XIII, gland cells squarish, arranged in transverse rows, midventrally absent. Head pore at 0/I, no dorsal pores. Epidermal gland cells inconspicuous in vivo. Thickness of body wall about 18–20 µm, and cuticle thin <1 µm.

Figure 1. 

Micrograph of Marionina puntaalanensis sp. nov. A. Entire specimen; B. Brain; C. Pharyngeal glands (marked with white arrows; spermathecae marked with black arrows); D. Anterior bifurcation of the dorsal vessel in III; E. Rounded coelomocytes; F. Lemon-shaped coelomocytes; G. Preclitellar nephridium; H, I. Sperm funnels (e = egg); J, K. Spermathecae (ectal glands marked with black arrows, ampullae marked with white arrows). A, C–J. in vivo, B, K. fixed, stained. Scale bars: 500 μm (A); 50 μm (B–D, F, H–J); 20 μm (E, G, K).

Brain (Fig. 1B) ca. 50–60 μm long (fixed), slightly longer than wide, incised posteriorly. Pharynx and postpharyngeal bulbs well developed. Prostomial ganglia absent. In the ventral nerve cord, perikarya continuous. First and second pharyngeal glands compact and united dorsally, in V with ventral lobes; the third pair free dorsally with elongate but stout ventral lobes (Fig. 1C). Chloragocytes from IV forming a denser layer from VI, about 15–20 μm long in vivo, filled with refractive globules. Transition between oesophagus and intestine gradual; oesophageal appendage and intestinal diverticula absent. Midgut pars tumida not seen. Dorsal vessel from clitellar region, blood colorless. The dorsal anterior blood vessel bifurcation in III (Fig. 1D). All coelomocytes nucleated oval, disc-shaped (Fig. 1E) or lemon-shaped (Fig. 1F) with granules, 13–20 μm long in vivo and 10–18 μm, fixed. Three pairs of preclitellar nephridia in 6/7–8/9, preseptal part consisting of funnel and coils of canals, postseptal part elongate, about two times as long as preseptal part, efferent duct terminal (Fig. 1G); the first postclitellar pair at 13/14 (mostly seven postclitellar pairs). Seminal vesicle absent or small, paired. Sperm funnels small, cylindrical, 100–140 μm long in vivo, 40–75 μm when fixed, and about 1.5–2.5 times longer than wide in vivo (1.5–2 times, when fixed), collar high and narrower than funnel body (Fig. 1, I). Spermatozoa 38–43 µm long, heads 15–22 µm in vivo and 20–32 µm long and heads 10–13 µm, when fixed. Sperm ducts short, about four times longer than the funnel, coiled into a loose spiral, diameter 7–10 µm in vivo and 4–5 µm, when fixed. Male copulatory organs small and compact, 25–36 µm long, 23–40 µm wide, and 15–30 μm high in vivo (22–30 µm long, 20–27 µm wide, and 25–30 µm high, when fixed). Small subneural glands in XIII–XIV (in one specimen absent). Ectal duct of spermatheca 24–38 µm long, surrounded along the length by glands and one larger, 15–27 µm long, sessile gland at orifice. Ampulla oval, 24–40 µm wide and 40–55 µm long in vivo (20–30 µm wide, 25–35 µm long, fixed), in the lumen with some sperm (Fig. 1J, K). Ampulla attached with a short ental duct to the oesophagus. One or two mature eggs at a time.

The new species is named after the Punta Ala beach, where it was found.

Known from Loc. 3 and Loc. 2., the intertidal zone is near Punta Ala (Grosseto) and Castiglione della Pescaia, Italy, in the decaying seagrass detritus.

Among the mostly intertidal small Marionina species with two chaetae in all chaetal bundles and without sperm rings in spermathecae, eight species are similar to the new species: M. istriae Giere, 1974; M. miniampullacea Shurova, 1978; M. magnifica Shurova, 1978; M. mica Finogenova, 1972; M. aberrans Finogenova, 1973; M. elgonensis Černosvitov, 1938; M. neroutsensis Coates, 1980; and M. mesopsamma Lasserre, 1964. The main differences are as follows: M. istriae is larger (body length 7–10 mm, segment number 38–43 vs. body length 2–2.5 mm, segment number 19–30), the chaetae are larger (65 µm long vs. 15–20 µm long), and a larger ectal gland is absent. M. miniampullacea is also larger (body length 4–5 mm), the chaetae are also larger (50 µm long), the dorsal vessel origin is in VII, and there is a rosette of glands at the orifice of the spermathecal duct. M. magnifica is larger (body length 4–5 mm), sometimes 3–4 chaetae occur, the chaetae are larger (40–50 µm long), and a larger ectal gland absent. M. mica has three ventral lobes of the pharyngeal glands in IV, V, and VI; the third pair is connected dorsally (vs. only in V; the third pair free); the dorsal vessel origin is in VIII; and the anterior blood vessel bifurcation is prostomial (also known as lumbricillinae-type). In M. aberrans, the cuticle is thick (2.5 µm vs. <1 µm), there is a large rosette of ectal glands, the preseptal part of the nephridia consists only of the funnel, and the sperm duct is long (vs. short). M. elgonensis is similarly small, but the ectal gland of the spermathecal ectal duct is absent. In M. neroutsensis, all pharyngeal glands are without ventral lobes; the ectal glands of spermathecae are absent, but a seminal vesicle is present (vs. absent). M. mesopsamma is larger (6 mm long), has a seminal vesicle, has only two pairs of preclitellar nephridia, and the spermathecae are free.

Holotype : Ma. 6, slide No. 3081. Type locality: (Loc. 3.) Italy, Punta Ala Grosseto, Castiglione della Pescaia, decaying seagrass detritus, 42°46'00.0"N, 10°51'31.0"E, Leg. András Dózsa-Farkas and Kinga Dózsa-Farkas, 24 Sep 2020.

Paratypes : in total, 15 specimens: P.147.1 slide No. 3027, P.147.2 slide No. 3049 (two specimens), P.147.3 slide No. 3050 (two specimens), P.147.4 slide No. 3053 (two specimens), P.147.5 slide No. 3058, P.147.6 slide No. 3060, P.147.7 slide No. 3071, P.147.8 slide No. 3072, P.147.9 slide No. 3074, P.147.10 slide No. 3075, P.147.11 slide No. 3079, P.147.12 slide No. 3080. Same data as for holotype.

20 specimens (10 only in vivo).

(1) Small size (body length 2.3–3.3 mm, 130–220 µm wide at clitellum, in vivo, segment number 17–22); (2) chaetae straight with ental hook, two chaetae per bundle, slightly longer at the posterior end of the body; (3) clitellum saddle-shaped; (4) brain truncate posteriorly; (5) first and secondary pharyngeal glands united dorsally with small ventral lobes; the third pair elongate, free dorsally; (6) dorsal vessel from clitellar region, blood colorless. The dorsal anterior blood vessel bifurcation anteriorly behind the pharynx; (7) two pairs of preclitellar nephridia; (8) coelomocytes oval or disc-shaped with granules, 14–22 μm long in vivo; (9) seminal vesicle well developed; (10) sperm funnel 1.5–3 times longer than wide in vivo, collar high and narrower than funnel body, spermatozoa 44–60 µm long, heads 20–25 µm in vivo; (11) male copulatory organ small and compact, 30–40 µm long in vivo; (12) small subneural glands are in XIII–XIV; (13) ectal duct of spermatheca short, surrounded by glands. Ampulla spherical, diameter 40–55 µm in vivo, the lumen characteristically full of many spherical sperm rolls. Ampulla attached to the oesophagus; (14) 1–3 mature eggs at a time.

Small species, holotype 2.1 mm long, 134 µm wide at VIII and 150 µm at clitellum (fixed), segment number 21. Body length 2.3–3.4 mm, width 110–188 µm at VIII and 130–220 µm at clitellum, in vivo, length of fixed specimens 1.1–2.1 mm, width 118–160 µm at VIII and 125–180 µm at clitellum, segment number 17–24. Chaetae straight with ental hook. Chaetal formula: 2 - 2: 2 - 2 (in one case, three chaetae were in one ventral bundle of the segment III). The chaetae are equal in size within the bundles, a little longer in the ventral bundles than in the lateral ones. Chaetae are 20–30 × 2.2 μm in preclitellar segments and 28–35 × 2.8–3 μm at the posterior end of the body (Fig. 2D). Clitellum saddle shaped in XII-1/2 XIII, gland cells squarish, arranged in about 16–17 transverse rows (Fig. 2C), midventrally absent (Fig. 2B). Head pore at 0/I, no dorsal pores. Epidermal gland cells inconspicuous in vivo. Thickness of body wall about 15–16 µm, and cuticle thin (1 µm).

Figure 2. 

Micrograph of Marionina orbifera sp. nov. A. Brain; B. Clitellar glands absent ventrally (the male copulatory glands = m); C. Clitellum (sperm funnels = sf); D. Chaetae at the body end; E. Anterior bifurcation of the dorsal vessel in III; F. Pharyngeal glands, lateral view (primer pharyngeal gland pairs marked with white arrows, ventral lobes marked with black arrows); G. Coelomocytes; H. Large seminal vesicle; I. Sperm funnels (sperm funnels = sf, seminal vesicle = sv); J. Subneural gland in XIV; K–M. Spermathecae (the sperm rolls in the ampullae marked with black arrows, the glands at the ectal duct marked with white arrows). A–E, G–I, K–M. In vivo; F, J. Fixed, stained. Scale bars: 50 μm (A, C, E, F, I, J, K); 20 μm (B, D, G, H, L, M).

Brain (Fig. 2A) ca. 80 μm long (fix.) slightly longer than wide, truncate posteriorly. Pharynx well developed. Prostomial glanglia absent. In the ventral nerve cord, perikarya continuous. First and secondary pharyngeal glands compact and united dorsally, with small ventral lobes; the third pair elongate and free dorsally (Fig. 2F). Chloragocytes from IV forming a denser layer from VI, about 17–30 μm long in vivo, filled with refractive globules. Transition between oesophagus and intestine gradual; oesophageal appendage and intestinal diverticula absent. Midgut pars tumida not seen. Dorsal vessel origin from clitellar region; blood colorless. The dorsal anterior blood vessel bifurcation in III (Fig. 2E). All coelomocytes nucleated oval or disc-shaped with granules, 14–22 μm long in vivo (Fig. 2G) and 8–10 μm fixed. Two pairs of preclitellar nephridia in 7/8 and 8/9, preseptal part consisting of funnel and coils of canal, postseptal part elongate, about four times as long as preseptal part, efferent duct terminal. The first postclitellar pair of nephridia at 13/14. Seminal vesicle well developed, paired, extending anteriorly to X or IX and posteriorly to XII–XIII (Fig. 2H). Sperm funnels cylindrical, 85–140 μm long in vivo, 60–100 μm, fixed, and about 1.2–3 times longer than wide, collar high, and narrower than funnel body (Fig. 2C, I). Spermatozoa 44–60 µm long (in two specimens they were 80–87 μm long), heads 20–25 µm in vivo (Fig. 2I), (23–28 µm and 10–15 µm, respectively, when fixed). Sperm ducts short, coiled into a loose spiral, diameter 5–7 µm, in vivo. Male copulatory organs small and compact (Fig. 2B), 30–40 µm long, 20–35 µm wide, and 15–30 μm high, in vivo (25–38 µm long, 18–25 µm wide, and 20–30 µm high, when fixed). Small subneural glands in XIII–XIV (Fig. 2J). The ectal duct of spermatheca short, 20–35 µm long, surrounded along the length by glands, somewhat larger entally, no distinct rosette around the orifice (Fig. 2M). Ampulla spherical, diameter 40–55 µm in vivo (33–40 µm, fixed), the lumen filled with many spherical sperm rolls (Fig. 2K–M). Ampulla attached with a short ental duct to the oesophagus. 1–3 mature eggs at a time.

The species is named after the characteristic sperm rolls („orb”) in the spermatheca [orbifera = orb-bearing (Latin)].

Known from the type locality, decaying seagrass detritus.

Among the intertidal small Marionina species, nine species (M. sjaelandica Nielsen & Christensen, 1961, M. levitheca Erséus, 1990, M. coatesae Erséus, 1990, M. swedmarki Lasserre & Erséus, 1976, M. vancouverensis Coates, 1980, M. limpida Shurova, 1979, M. cana Marcus, 1965, M. transunita Coates, 1990, M. southerni (Černosvitov, 1937) and the new species are characterized by spherical sperm rolls in the spermathecal ampulla. The main differences are as follows: The spermathecae of M. sjaelandica and M. coatesae are similar to the new species; more sperm rolls are in the spermathecae, but not in the cavity, but embedded in the walls of the ampulla. Both species have more segments (segment number 24–27 in M. sjaelandica, 27–31 in M. coatesae, vs. 18–24 segments in the new species). M. levitheca is larger (segment number 38–41), the sperm rolls are arranged in distinct globular cavities scattered in the wall, and there are no glands at the ectal duct of the spermatheca. In M. swedmarki, the spermathecal orifice has a conspicuous gland-rosette. M. vancouverensis has a maximum of six chaetae per bundle (vs. only two in the new species). M. limpida is larger (6–8 mm long, vs. 2.3–3.3 mm), the subneural glands are only in XIII (vs. XIV–XV), the sperm funnel and sperm duct are longer, and the brain is incised posteriorly. In M. cana, the sperm rolls are in the walls of the ampulla, the ectal duct is not glandular, and the dorsal vessel origin is in IX, not in the clitellar region; moreover, the brain is incised posteriorly. M. transunita is also larger (with segment numbers 26–40), and the two spermathecae are connected entally. M. southerni is 8–10 mm long with 28–36 segments; the coelomocytes are black in transmitted light; and the spermatheca has many sessile diverticula.

Holotype : Ma. 7, slide No. 3128. Type locality: (Loc. 4) Hungary, Lake Balaton, Bélatelep, Strand Bátori, lake shore, wet sand between the roots of willow trees, 46°43'51.5"N, 17°31'41.7"E, Leg. György Reichart, 14 Feb 2021.

Paratypes : In total, 22 (20 adult and 2 subadult) specimens: P.148.1 slide No. 3116 (three specimens), P.148.2 slide No. 3117 (three specimens), P.148.3 slide No. 3122 (two specimens), P.148.4 slide No. 3123 (four specimens), P.148.5 slide No. 3124 (two specimens), P.148.6 slide No. 3126 (two specimens), P.148.7 slide No. 3127, P.148.8 slide No. 3129 (two specimens), and P.148.9 slide No. 3130 (three specimens). Same data as for holotype.

19 specimens (10 investigated only in vivo) + three specimens for DNA analysis.

(1) Small size (body length 2–3.3 mm, 137–190 µm wide at clitellum, in vivo), segment number 19–29; (2) maximum five chaetae per bundle, chaetae straight with ental hook and mostly not equal in size; (3) clitellum saddle-shaped; (4) first and second pairs of pharyngeal glands compact and united dorsally without ventral lobes; the third pair free and elongated; (5) transition between oesophagus and intestine gradual, midgut pars tumida in XVIII–XXII, extending over 3–4 segment lengths; (6) dorsal vessel origin in XII, blood colorless, the dorsal anterior blood vessel bifurcation anteriorly behind the pharynx; (7) three pairs of preclitellar nephridia; (8) coelomocytes nucleated oval or lemon-shaped with fine granules, 15–24 μm long in vivo; (9) seminal vesicle absent; sperm morulae occur in all segments; (10) sperm funnel 1.5–3 times longer than wide in vivo, collar high and narrower than funnel body; spermatozoa 46–70 µm long, heads 20–30 µm in vivo; (11) male copulatory organ oval or bean-shaped and compact, 33–47 µm long in vivo; (12) ectal duct of spermatheca surrounded by glands, wider proximally; one larger sessile gland at orifice; ampulla globular, diameter 29–40 µm in vivo with some sperm-threads or sperm bundle in it; ampulla attached to oesophagus.

Small species (Fig. 3A), holotype 2.5 mm long, 115 µm wide at VIII and 138 µm at clitellum (fixed), segment number 28. Body length 2.0–3.3 mm, width 130–160 µm at VIII and 137–190 µm at clitellum in vivo, length of fixed specimens 1.5–2.5 mm, width 90–135 µm at VIII and 115–160 µm at clitellum, segment number 19–29. Chaetae straight with ental hook (Fig. 3D, E). Chaetal formula: 2.3,(4) - 2.3: (3)4.5,(6) - 2.3,(4). The chaetae are not exactly equal in size within the bundles; often the middle chaetae slightly smaller than the ental ones, e.g., in the ventral preclitellar bundles, 20–19.5–18–20 µm or 25–22–20.4–24.2 µm long and 1.6–1.9 µm wide. The ventral chaetae slightly longer than lateral ones. At the posterior end of the body, chaetae are 21–27 μm long. Clitellum saddle-shaped in XII-1/2 XIII, gland cells squarish, arranged in about 15–18 transverse rows (Fig. 3C), midventrally absent. Head pore not seen, no dorsal pores. Epidermal gland cells inconspicuous in vivo. Thickness of body wall about 10–13 µm; cuticle thin (<1 µm).

Figure 3. 

Micrograph of Marionina reicharti sp. nov. A. Entire specimen (e = egg); B. Brain; C. Clitellar glands, dorsal view; D. Chaetae, anterior bifurcation of the dorsal vessel in III, ventral view; E. Chaetae posteriorly; F. Pharyngeal glands (spermathecae marked with arrows); G. Chloragogen cells; H. Preclitellar nephridium; I. Coelomocytes; J. Pars tumida of midgut in XX–XXI. A–I. In vivo; J. Fixed, stained. Scale bars: 200 μm (A); 50 μm (F–H); 20 μm (B–E, G, I, J).

Brain (Fig. 3B) ca. 47–51 μm long (fixed), slightly longer than wide, incised posteriorly. Prostomial glanglia absent. In the ventral nerve cord, perikarya continuous. First and second pair of pharyngeal glands compact and united dorsally without ventral lobes; the third pair free and elongate (Figs 3F, 4G). Chloragocytes from IV forming a denser layer from VI, about 8–12 μm long in vivo, filled with refractive globules (Fig. 3G). Transition between oesophagus and intestine gradual; oesophageal appendage and intestinal diverticula absent. Midgut pars tumida in XVIII–XXII, extending over 3–4 segment lengths (Fig. 3J). Dorsal vessel from XII, blood colorless. The dorsal anterior blood vessel bifurcation in III. All coelomocytes nucleated oval or lemon-shaped with fine granules, 15–24 μm long in vivo (Fig. 3I) and 8–10 μm, fixed. Three pairs of preclitellar nephridia in 6/7–8/9, preseptal part consisting of funnel and coils of canal, postseptal part elongate, about 1.7–3 times longer than the preseptal part, efferent duct terminal (Fig. 3H). Seminal vesicle absent, sperm morulae and sperm bundles may occur in all segments (Fig. 4A). Sperm funnels cylindrical, 60–95 μm long in vivo, 40–70 μm, fixed, and about 1.5–3 times longer than wide in vivo (1.2–2 times, when fixed), collar high, and narrower than funnel body (Fig. 4D–F). Spermatozoa 46–70 µm long, heads 20–30 µm in vivo, 30–50 µm long, heads 10–22 µm, when fixed. Sperm ducts short, about 2.5–3.5 times longer than the funnel, coiled into a loose spiral, diameter 7–9 µm, in vivo. Male copulatory organ oval or mostly bean-shaped and compact (Fig. 4B, C, F), 33–47 µm long, 18–26 µm wide, and 18–26 μm high, in vivo (30–40 µm long, 21–28 µm wide, and 18–25 µm high, when fixed). Subneural glands absent. Ectal duct of spermatheca, 26–38 µm long, surrounded along the length by glands, 14–18 µm wide proximally and 10–14 µm wide distally in vivo (20–23 µm long, 14–20 µm wide proximally, and 12–16 µm wide distally, when fixed). One larger, 15–22 µm long, sessile gland at orifice in vivo (15–20 µm, fixed) (Fig. 4G–J). Ampulla globular, diameter 29–40 µm in vivo (25–40 µm, fixed), lumen with some sperm-threads (Fig. 4H) or 1–3 sperm bundles (Fig. 4I). Ampulla attached with a short ental duct to the oesophagus. One or two mature eggs at a time (Fig. 4C, F).

Figure 4. 

Micrograph of Marionina reicharti sp. nov. A. Sperm bundles in the coelom; B, C. Male copulatory organs (e = egg, sd = sperm duct); D–F. Sperm funnels (male copulatory organs marked with a white arrow); G–J. Spermathecae (ectal glands marked with black arrows, ampulla marked with white arrows, in H. Sperm thread in ampulla, in I. Sperm bundles in ampulla). A–C, E–I. In vivo; D, J. Fixed, stained. Scale bars: 50 μm (C, F, G); 20 μm (A, B, D, E, H–J).

The new species is named in the honor of György Reichart, who collected the sample with this species.

Known from the lake shore of Lake Balaton at Bélatelep, Strand Bátori, Hungary, in wet sand between the roots of willow trees (Loc. 4). Earlier, they were identified as Marionina spicula (Leuckart, 1847) at four stations of the Lake Balaton (between Fűzfő and Alsóörs, Balatonberény, and Bélatelep) in a fauna investigation in 1990–1992 (Dózsa-Farkas 1995). Unfortunately, these specimens were lost.

Some small enchytraeid worms during the former study of Lake Balaton shore fauna were identified as Marionina spicula (Dózsa-Farkas 1995); therefore, the question was raised if they really belonged to this species known typically from marine habitats. However, in two papers (Lafont and Juget 1976; Rodriquez 1986), the species was observed along rivers without a detailed morphological description, and its euryhalinity was also reported previously (Giere 1971). This initiated our study with a freshly collected sample from the shore of Lake Balaton, combining DNA sequencing with morphological investigation. Since some specimens fitting the description of M. spicula (Nielsen and Christensen 1959) were detected in the Adriatic shore samples (collected in 2019), those specimens were also included in our comparison. Furthermore, specimens collected from a Danish seashore in 1999 (site 5) were also studied, but unfortunately, we were not able to obtain DNA sequences from them due to their fixation in Bouin’s fluid. The results of the molecular analysis revealed that the DNA sequences of the Hungarian specimens (Lake Balaton) differ from those of the individuals collected by us from the Adriatic seashore (site 1, Table 1) and from the single DNA sequence of M. spicula in NCBI GenBank (see details below), so there is support at the DNA level that the Balaton specimens belong to a new species.

Our species comparison is based on Nielsen and Christensen (1959), and it includes observations on living individuals collected in Denmark on the Nivå coast in 1999 (site 5) and reinvestigated (as fixed material) in this study. The morphological differences are as follows: M. reicharti sp. nov. is smaller (2–3.3 mm length vs. 4–5 mm length in M. spicula in Denmark, in vivo, 1.5–2.5 mm length vs. 2.1–2.6 mm length, fixed), segment number 19–29 vs. 27–30. Sperm funnel slightly shorter (40–70 µm long vs. 67–90 µm, fixed), the spermatozoa are longer in the new species (30–50 µm, heads 11–22 µm vs. 20–38 µm, heads 10–17 µm in M. spicula). The ectal duct of the spermatheca is longer in Danish M. spicula specimens and proximally wider (20–50 µm long and 16–50 µm wide vs. 20–23 µm long and 14–20 µm wide in the new species). Origin of the dorsal vessel from segment XII vs. from XIII in Danish M. spicula specimens.

Keeping in mind that the Mediterranean M. spicula specimens studied here may belong to a different species, and since the origin of M. spicula specimen CE2561 is from Sweden (see additional details below), we performed a morphological comparison of Mediterranean M. spicula with the new species: M. reicharti sp. nov. is smaller and has in general fewer segments: length 3 mm, width 137–190 μm at clitellum in vivo, 19–29 segments (vs. 3–7 mm, 200–430 μm, 21–39 segments, respectively). The maximum number of ventral chaetae is lower, 5–6 (vs. 7–8). The clitellum is saddle shaped (vs. ring-shaped, glands absent only between the male openings). The spermatozoa are 46–70 µm long, heads 20–30 µm in vivo (vs. 60–120 µm long and 30–43 µm), the male copulatory organ is also smaller, 33–47 µm long, 18–26 µm wide, 18–26 µm high (vs. 45–70 × 45–60 × 35–50 µm). Furthermore, the size of the spermatheca is different: ectal duct 26–38 long, ectal gland 15–22 µm long, diameter of ampulla 29–40 µm in vivo (vs. 35–51 µm, 30–40 µm and 40–63 µm, respectively). The coelomocytes and the size of sperm funnels are not comparable, because these traits in the specimens of M. spicula collected at the Adriatic seashore are very variable (a sign of that possibly M. spicula is a species complex).

Morphological notes on the Marionina spicula (Leuckart, 1847) specimens of the Adriatic seashore

Figs 5–6

Material examined. About 50 specimens were investigated in vivo, slides were made from 28 specimens, and 11 specimens were used for DNA analysis. Collecting site: (Loc. 1) Croatia, Istria, Kale Cove seashore, Adriatic Sea, Kamenjak Peninsula, decaying seagrass (Zostera) detritus, 44°51'13.0"N, 13°58'50.5"E, Leg. Júlia Török, 03 Apr 2019, and 05 Sep 2020.

Description of new material. Small worms, body length 3–7 mm, width 200–430 µm at clitellum in vivo; length of fixed specimens 1.9–3.6 mm, width 170–320 μm at clitellum, segment number 21–39. Chaetae straight with ental hook. Chaetal formula variable: 2-5 - 4-2: 4-8 - 6-2. The chaetae unequal in size within the bundles. Mostly the chaetae towards the midlines of the body are shorter than the lateral ones, or the chaetae in the middle of the bundles are shorter (Fig. 5A–C). Clitellum ring-shaped in XII-1/2 XIII, gland cells arranged in irregular transverse rows (Fig. 5E), between the male openings absent (Fig. 5F). Head pore at the middle of prostomium, no dorsal pores. Epidermal gland cells inconspicuous in vivo.

Figure 5. 

Micrograph of Marionina spicula. A. Chaetae maximum 5 in a ventral bundle; B. The inner chaetae shorter; C. Chaetae maximum 7-8 in a ventral bundle. A, B, and C from different specimens; D. Brain; E. Clitellar glands, dorsal view; F. Clitellar glands absent between the male copulatory organs; G. Anterior bifurcation of the dorsal vessel in III (spermathecae marked with white arrows); H. Lighter coelomocytes; I. Dark coelomocytes; J. Coelomocytes with fewer granules; K. Coelomocytes full with granules. All pictures are in vivo. Scale bars: 50 μm.

Brain (Fig. 5D) 62–87 μm long (fixed), 1.5 times longer than wide, incised posteriorly. Pharynx and postpharyngeal bulbs well developed. Prostomial ganglia absent. In the ventral nerve cord, perikarya continuous. First and secondary pharyngeal glands compact and united dorsally; the third pair free (Figs 5G, 6F). Chloragocytes from IV forming a denser layer from VI, about 15–20 μm long in vivo, filled with refractive globules. Transition between oesophagus and intestine gradual; oesophageal appendages and intestinal diverticula absent. Midgut pars tumida not seen. Dorsal vessel from XII, blood colorless. The dorsal anterior blood vessel bifurcation in III, pharyngeal (Fig. 5G). Coelomocytes variable (Fig. 5H–K), nucleated, disc-shaped with gray granules; in some specimens, the coelomocytes are with few granules (Fig. 5J), so they are pale in the coelom. In other specimens, the coelomocytes are filled with granules (Fig. 5K), and the coelomocytes are in such large numbers that they fill the entire coelom and are dark gray in transmitted light (Fig. 5I), 15–24 μm long in vivo, and 15–17 μm, fixed. Three pairs of preclitellar nephridia in 6/7–8/9, preseptal part consisting of funnel and coils of canal, postseptal part elongate, about 2–2.5 times longer than the preseptal part, efferent duct terminal (Fig. 6A). Seminal vesicle absent. Sperm funnels cylindrical, very variable; they can be about 110–150 μm long and 1.7–2.4 times longer than wide (Fig. 6B, C); in other specimens, they are very large, 170–300 μm long and 3–4.5 times longer than wide in vivo (Fig. 6D, E), collar 10–25 μm high and narrower than funnel body. Spermatozoa 60–120 µm long, heads 30–43 µm in vivo. Sperm ducts short, about three times longer than the funnel, diameter 10–13 µm, in vivo (Fig. 6C). Male copulatory organs compact (Fig. 5F), 45–70 µm long, 45–60 µm wide, and 35–50 μm high in vivo (32–58 µm long, 30–53 µm wide, and 28–40 µm high, when fixed). Ectal duct of spermatheca 35–51 µm long, surrounded along the length by glands and one larger, 30–40 µm long, sessile gland at orifice. Ampulla rounded (diameter 40–63 µm wide in vivo, 35–53 µm, fixed), sometimes with a rather thick wall (6–14 µm) in the lumen with sperm (Fig. 6F, G). Ampulla attached with a short ental duct to the oesophagus. One or occasionally two mature eggs at a time (Fig. 6C). In this species, as already pointed out by Giere (1971), it is often observed that the coelomic fluid, released through the anus, attaches itself to the grains of sand (Fig. 6H).

Figure 6. 

Micrograph of Marionina spicula. A. Nephridium; B. Shorther sperm funnels; C. Shorther sperm funnels (marked with arrows) with a short sperm duct (e = egg); D, E. Large sperm funnels; F–G. Spermathecae (ampullae marked with arrows); H. Body-end with ejected sticky mucus. A–F, H. in vivo, G. fixed, stained. Scale bars: 50 μm (A–F, H); 20 μm (G).