Clayey soil, meadow (site 9), Yongnuni-orum, Gujwa-eup, Jeju Island, South Korea.

NIBRIV0000813658, slide No. 2329, adult, stained whole mounted specimen, collected on 26 Oct 2016 by Y. Hong. Paratypes. In total six stained adult and one subadult specimens on slides, coll. Y. Hong. NIBRIV0000813659, slide No. 2459 and NIBRIV0000813660, slide No. 2462 from type locality. P.120.1–P.120.4, slides No. 2305, 2460, 2478, 2482 from type locality. P.120.5, subadult specimen, slide No. 2464, site 8 (clayey soil at the bottom of the dormant crater, meadow; 33.45859°N; 126.83192°E; 193 m asl.), 26 Oct 2016. Further material examined. Two specimens for DNA analysis and four subadults and six juvenile specimens only in vivo.

The new species can be recognized by the following combination of characters: (1) small, slender worms (2.5–4.2 mm long and 160–220 μm wide at clitellum in vivo), segments 25–31; (2) six pyriform glands per segment in general; (3) clitellum weakly developed, interrupted middorsally and midventrally, with two elongate, “baguette-like” packages of gland cells on each dorso-lateral side; (4) dorsal blood vessel from VII; (5) pharyngeal glands at 4/5–6/7 connected dorsally, with ventral lobes and no secondary glands; (6) two pairs of preclitellar nephridia; (7) pars tumida of midgut from XII–XVI, extending over 2–3 segments, circumferal; (8) sperm funnels small, barrel-shaped, collar well developed about as wide as funnel body; (9) male pores in XI, ventro-lateral, each pore surrounded by small inconspicuous glands; (10) spermathecae free, confined to V with an asymmetrical dilation of ampulla and the ental tube ending in an oval reservoir.

Small, slender worm (Fig. 2E). Holotype (fixed) 3.2 mm long, 190 µm wide at VIII and 200 µm wide at clitellum (fixed), 31 segments. Paratypes 2.5–4.2 mm long, 155–200 µm wide at VIII and 160–220 µm wide at clitellum in vivo; 2.4–3.6 mm long, 150–210 µm wide at VIII and 160–210 µm wide at clitellum when fixed; segments 25–31. Head pore on prostomium (Fig. 3A). Clitellum in XI–1/2 XII weakly developed, gland cells absent dorsally and ventrally, laterally cells in transverse rows (Figs 1C, 2G, H). On each side dorso-laterally two elongate, baguette-like packages of swollen gland cells (in the middle hyalocytes, on the two margins granulocytes) narrowing at both ends (Figs 1C, 2G, I), length of baguette 190–270 μm and width 21–26 μm in the middle in vivo (100–180 μm and 20–25 μm in fixed specimens, respectively). Spermathecal pores at 4/5 in lateral position. Male pores in XI (Fig. 2H).

Figure 1. 

A–D Achaeta multisacculata sp. n.: A Spermatheca B Sperm funnel C Clitellar glands, lateral view (glands middorsally and midventrally absent; two “baguette-like” packages of gland cells dorso-laterally, granular gland cells in transverse rows latero-ventrally) D Brain E–G Fridericia floriformis sp. n.: E Sperm funnel F Coelomocytes G Spermatheca.

Figure 2. 

Micrograph of Achaeta multisacculata sp. n. A Head lateral view (b = brain, knob on brain marked with black arrow, first dorsal pyriform glands marked with white arrow) B Brain dorsal view (knob on brain marked with arrow) C Cuticle thicker dorsally than ventrally, lateral view D Transverse body wall striation by strong ring muscles E Forepart of body to VII, lateral view (b = brain, ph = pharynx, oe = oesophageal appendages, dorsal pyriform glands marked with black arrows, lateral pyriform glands marked with wider white arrows, ventral pyriform glands marked with narrower white arrows) F Pyriform glands in IV–IX lateral view G Clitellar glands of holotype, lateral view (dorso-laterally 2 elongate, “baguette-like” packages of hyalocytes marked with black arrows, granulocytes ventro-laterally marked with white arrow) H Granular clitellar glands in transverse rows ventrally, lateral view (male openings marked with arrow) I Two baguette-like packages of clitellar glands (marked with arrows, in the middle hyalocytes, on the margins granulocytes) J Segments III–VIII, lateral view (oe = oesophageal appendages with meandering canal, marked with black arrow, dv = origin of dorsal vessel, n = first nephridium, ganglia of ventral nerve cords marked with white arrows) K Segments IV–VIII of paratypes NIBRIV0000813659, No. 2459 lateral view (p = pharyngeal glands, oe = oesophageal glands, spermatheca marked with arrow) A, D–F, H–J in vivo, B–C, G, K fixed, stained. Scale bars: 50 μm, in H, I: 20 μm.

Body wall in vivo 10–21 μm with cuticle 5–9 μm thick dorsally and 3–5 μm thick ventrally (Fig. 2C). Ring muscles strong, resulting in transverse body wall striation (Fig. 2D). Septa 4/5–7/8 thickened (Fig. 2K). Frontal prostomial epithelium thickened ventrally. Pyriform epidermal glands (Fig. 2E, F) generally 3 pairs in dorsal, lateral and ventral position in each segment (XI also), sometimes difficult to observe or lateral, and ventral pairs absent: size variable, dorsal pairs largest, from II onwards, length 17–18 µm at II, 22–40 µm preclitellarly, 26–54 µm in the middle of body 22–26 µm posteriorly in vivo: lateral and ventral pairs from III onwards, length in vivo 11–25 µm and 11–18 µm preclitellarly, 21–24 and 15–17 µm in the middle of body, 19–25 and 12–20 µm posteriorly, respectively, but size subequal when fixed.

Brain posteriorly rounded, anteriorly convex with a conspicuous knob, 77–90 μm long, 1.6–1.8 times longer than wide in vivo (Figs 1D, 2A) (70–95 μm long and 1.5 times longer than wide, fixed, Fig. 2B). Suboesophageal ganglion of ventral nerve cord in II–IV undivided, posterior ganglia segmental and separate (Fig. 2J). Two small paired post-pharyngeal bulbs present. All pharyngeal glands at 4/5–6/7 united dorsally and with ventral lobes (Fig. 2K): first pair of glands largest, no secondary glands. Two pairs of preclitellar nephridia at 7/8–8/9 slightly constricted by septa: length ratio anteseptale : postseptale 1 : 2–3 preclitellarly, postseptale bent and tapering gradually into efferent duct, with small terminal vesicle (Fig. 3B). About 6 pairs of postclitellar nephridia (Fig. 3C) from 19/20. Dorsal blood vessel from VII (Fig. 2J), often with intensive pulsation in VII and VI, blood colourless. Coelomocytes disc-like, with fine granules, dark brown with clear nucleus, about 15–30 μm long in vivo (Fig. 3E, F) (12–16 μm, fixed). One pair of oesophageal appendages well developed dorso-laterally in V, with meandering canal in IV (Fig. 2E, J, K), clearly visible only in live worms (Fig. 3D). Chloragocytes brown, about 10–13 μm long in vivo. Midgut pars tumida inconspicuous, circumferal (i.e., not confined to ventral region of intestine), in XII–XVI (occupying 2–3 segments). Pygidium short, anal muscles developed (Fig. 3G).

Figure 3. 

Micrograph of Achaeta multisacculata sp. n. A Head pore dorsal view (marked with arrow) B Preclitellar nephridia at 8/9 (marked with arrow) C Last nephridia at 26/27 of paratype P.120.2, No. 2460 D Oesophageal appendages in V (marked with black arrow), meandering canal in IV (marked with white arrow) E–F Coelomocytes G Pygidium, the anal muscles well developed H–J Sperm funnels K–L Spermathecae of paratype NIBRIV0000813659, No. 2459 (the diverticulum-like dilation of ampulla marked with arrow in L) M Spermatheca of paratype P 120.3 slide No. 2478 (here the ental reservoir bent back into IV marked with arrow). B, D–E, G, I, J in vivo A, C, F, H, J–L fixed, stained. Scale bars: 50 μm, in H, I: 20 μm.

Sperm funnels small, mostly barrel-shaped, 42–65 μm long in vivo (26–42 μm, fixed), about 1.5–2 times longer than wide, collar distinct 8–10 μm high, about as wide as diameter of funnel body (Figs 1B, 3H–J). Sperm ducts about 6 μm thick in vivo (4–5 μm, fixed). Spermatozoa with unusual strong tails (Fig. 3H), 50–70 μm long, heads 15–22 μm long in vivo (26–42 μm and 11–14 μm, fixed). Seminal vesicle absent. Male copulatory organs small oval, widely separated ventro-laterally, pore surrounded by small inconspicuous glands (Fig. 2H). Spermathecae free, confined to V (in one case extending into VI in vivo and in one case bent backwards into IV (slide 2478) (Fig. 3M). Ectal ducts 30–32 μm long and 14–16 μm wide in vivo (31–42 μm long and 9–10 μm wide, fixed), ducts slightly widen out to a dilation of ampullae with a diverticulum-like protrusion (dilation diameter 20–25 μm). After dilation, ental tubes (about 40–65 μm long and 20 μm wide) end in an elongated reservoir (24–45 μm long, 15–26 μm wide in vivo) (20–27 μm long and 13–18 μm wide, fixed) (Figs 1A, 3K–M).

Although the specimens are adult, the clitellar glands appear weakly developed. The reason is that this organ is fully developed only just before the release of an egg (as was remarked by Schmelz et al. 2008), and indeed our worms did not have mature eggs.

Named after the high number of ‘pyriform glands’ (sacculus = saccule, Latin).

Sequences deposited in GenBank: MH128727-MH128728 (ITS), MH124584-MH124585 (CO1).

In South Korea, at sites 8–9, Jeju Island, Yongnuni-orums, clayey soil, meadows.

Two Achaeta species with six pyriform glands per segment have been previously described: the European Achaeta aberrans Nielsen & Christensen, 1961 and the South American Achaeta piti Bittencourt, 1974, emended Schmelz et al. 2008. The new species can be easily distinguished from A. aberrans which has fewer segments, 20–23 (vs. 26–31 in the new species), dorsal vessel originating in VI (vs. in VII), oesophageal appendages small and only in V (vs. well developed in IV–V), male opening in XII (vs. in XI), coelomocytes oval, finely granulated and at one end tapering into a thin process (vs. discoid and brown), the preclitellar nephridia in 6/7 and 7/8 (vs. 7/8 and 8/9), the spermathecae, when present, with laterally symmetrical ampullae. The other species, A. piti is very similar to A. multisacculata sp. n., because of the reproductive organs (except the spermathecae) shifted one segment forward, the oesophageal appendages well developed with canal in IV-V, two pairs of nephridia in 7/8–8/9, dorsal blood vessel origin in VII and the spermatheca with ectal asymmetry. In addition, in both species two elongate, dorso-lateral „baguette-like” packages of hyalocytes occur in the clitellum. Characters that differentiate A. multisacculata sp. n. from A. piti are: (1) body size slightly smaller: 2.5–4.2 mm in vivo and 2.2–3.6 mm, fixed, 160–220 μm wide at clitellum [according to Schmelz et al. 2008, live A. piti worms are ca. 5 mm long and 150 μm wide in vivo (fixed type specimens are 3.5–5 mm long, 190 μm wide and specimens at Zoological Museum in Hamburg even longer, 4.5–6.5 mm and up to 250 μm wide)]; (2) segment number smaller, 26–31 (vs. 31–36); (3) brain anteriorly with a conspicuous knob (vs. without knob); (4) coelomocytes dark brown, 15–30 μm long (vs. pale, 15–18 μm long, cells somewhat darker than coelom); (5) sperm funnel smaller, mostly barrel-shaped, without vesicles, 42–65 μm long in vivo, 26–42 μm, fixed, only 1.5–2 times longer than wide (vs. cylindrical, with large vesicles, ca. half as long as body diameter, more than 3 times longer than wide); (6) pars tumida of midgut at XII–XVI, 2–3 segments long (vs. XX–XXIV); (7) in pygidium anal muscles clearly visible (vs. not strongly developed); (8) spermatheca similar but except one specimen always confined in V (vs. ampulla extending into VI or VII).

Clayey soil, meadow (site 9), Yongnuni-orums, Gujwa-eup, Jeju Island, South Korea.

NIBRIV0000813661, slide No. 2437, adult, not stained, whole mounted specimen, collected on 26 Oct 2016 by Y. Hong.

In total 18 adult stained and not stained specimens on slides and eight specimens in 70% ethanol, coll. Y. Hong. NIBRIV0000813662, slide No. 2293, DNA 1133, adult stained, whole mounted specimen from type locality. NIBRIV0000813663, slide No. 2427, from site 8 (clayey soil at the bottom of the dormant crater, meadow; 33.45859°N; 126.83192°E; 193 m asl.), 26 Oct 2016. P.121.1-P.121.14, slides No. 2291, 2314, 2332–2333, 2389, 2428–2432, 2436, 2438, 2440, 2481 from type locality. P.121.15–121.17, slides No. 2295, 2434, 2439 from site 8 (four specimens: slide 2434, 2436, 2438 and 2481 were not stained). P.121.18, five specimens in 70 % ethanol from type locality; and P.121.19, three specimens in 70 % ethanol from site 8.

Four juvenile and five adult specimens only in vivo (one of the whole, adult specimens was processed with molecular analysis, DNA 1136). One additional specimen in vivo and for molecular analysis (DNA 1088) from Mt. Hallasan, Jeju Island (Gwaneumsa trail, 33.41667°N, 126.55000°E, 634 m asl., 26 Oct 2016, coll. Y. Hong), referred as ‘Fridericia sp. 2’ in Dózsa-Farkas et al. (2018).

The new species can be recognized by the following combination of characters: (1) large size (body length 14–20.5 mm in vivo), segments 48–65; (2) lateral chaetae often absent, maximum 2 per bundle, ventrally maximum 3–4 chaetae per bundle; (3) clitellum well developed, between bursal slits and before the male apparati only granulocytes; (4) body wall strong and cuticle thick (3–5 μm); (5) five preclitellar pairs of nephridia; (6) coelomo-mucocytes c-type occasionally with some refractile vesicles, lenticytes scarce and small; (7) dorsal vessel from XV–XVIII; (8) chylus cells in XII–XV, occupying 2–3 segments; (9) bursal slit longitudinal slightly bent, with small transverse extensions; (10) seminal vesicle not brown; (11) subneural glands absent; (12) sperm funnel approximately as long as half body diameter, collar narrower than funnel diameter, spermatozoa 400–580 μm long, heads 100–150 μm in vivo; (13) spermatheca with 9–12 sessile diverticula of varying size mostly without sperm in them, ectal duct long without ectal glands and ampulla entally openings separately into oesophagus.

Large, whitish, stiff worms. Holotype 15.3 mm long, 470 μm wide at VIII and 550 μm at the clitellum (fixed), 59 segments. Body length of the paratypes 14–20.5 mm, width 400–530 μm at VIII and 500–640 μm at the clitellum in vivo. Length of fixed specimens 8–17.3 mm, width 470–580 μm at VIII and 500–620 μm at the clitellum. Segments 48–65. Chaetal formula: 1,2,(0) – 2,0,1,2 : 2,3,4 –(4),3,2,1. The inner chaetae being shorter and thinner than the outers: 30–35 × 2.5–3 μm and 54–63 × 5–6 μm (in preclitellar bundles). In the bundles with 2 chaetae the length of chaetae is different, in those with 3 chaetae one chaeta longer and the other two shorter. After the clitellum in lateral bundles of the middle part of body the chaetae mostly absent but at posterior body-end again occur 1 or 2 chaetae per bundle, length about 59–63 × 4.5–7 μm. Head pore at 0/I. Dorsal pores from VII; 2–3 transverse rows of hyaline epidermal gland cells per segment and in addition more transverse rows of dark yellow glands (visible only in vivo) (Fig. 4C). Clitellum in XII–1/2XIII, well developed, girdle-shaped, hyalocytes and granulocytes arranged in indefinite rows or reticulate pattern (Fig. 4E, F), between bursal slits and before the male apparati only granulocytes (Fig. 4G). Body wall strong, thickness about 40–54 μm, cuticle thick about 3–5 μm in vivo and fixed (Fig. 4I, K), in forepart slightly stronger than at the body end.

Figure 4. 

Micrograph of Fridericia floriformis sp. n. A–B Brain (B paratype P. 121.4, slide 2389) C Epidermal glands D Chylus cells in XII E–F Clitellar glands dorsal view G Clitellar glands ventrally, male copulatory organs of paratype P.121.7 slide No. 2429 (marked with arrows) H–I Coelomocytes J Oesophageal appendage K Body wall with strong longitudinal muscles and cuticle L–M Pharyngeal glands (L paratype P. 121.15 slide No. 2295 M paratype P121.8 slide No. 2430 dorsal vessel marked with arrows) N Pygidium with well-developed anal muscle, paratype 121.12 slide No. 2438. A, C–E, H, J in vivo B, G, I, K–M fixed, stained F, N fixed, not stained. Scale bars: 50 μm, in H: 20 μm.

Brain egg-shaped, about 140–180 μm long, about 1.5–2 times longer than wide in vivo (Fig. 4A) and 120–150 μm long and 1.4–1.7 times longer than wide in the fixed specimens (Fig. 4B). Oesophageal appendages long with many branches at the end in V (Fig. 4J). All pharyngeal glands with ventral lobes, those in 4/5 united dorsally, those in the 5/6 weakly united or unconnected dorsally and those in 6/7 unconnected dorsally but occasionally weakly united (Fig. 4L, M). All septa at 5/6–9/10 thickened. At anal region the radial gut dilator muscles well developed (Fig. 4N). Chloragocytes from V, 12–26 μm long in vivo. Dorsal vessel from XV–XVIII, (in one case in XIX), blood colourless. Midgut pars tumida in XXVI–XXXII occupying 4–7 segments (only in two specimens were visible). Five pairs of preclitellar nephridia from 6/7 to 10/11, length ratio anteseptale : postseptale 1 : 2–2.5, adseptal origin of the efferent duct. Coelomo-mucocytes c-type, rounded or elliptic, sometimes with some refractile vesicles, length 28–44 μm in vivo (Figs 1F, 4H), in the fixed worms with granules and 15–28 μm long (Fig. 4I). Lenticytes scarce, small 4–7 μm long. Chylus cells in XII–XV, occupying 2–3 segments (Fig. 4D).

Seminal vesicle in XI, not brown. Sperm funnels cylindrical (Figs 1E, 5A), about 180–330 μm long and about 2 times longer than wide (in vivo). Funnel length in fixed specimens 100–220 μm, funnel body 1.2–1.8 times longer than wide (Fig. 5B); collar narrower than funnel body. The length of spermatozoa 400–580 μm, heads 100–150 μm in vivo (Fig. 5A), in fixed specimens spermatozoa 200–360 μm long and sperm heads 70–80 μm. Diameter of sperm ducts 9–10 μm in vivo, (7.5–8 μm, fixed). Male copulatory organs 130–170 μm long, 60–140 μm wide and 70–80 μm high, fixed (Figs 4G, 5C–D), retractor muscles conspicuous (Fig. 5C). Bursal slits longitudinal, slightly bent, with small additional transverse extensions (Fig. 5E). Subneural glands absent. Spermathecae (Figs 1G, 5F–J): no ectal gland, ectal ducts long, about 360–500 μm and 20–25 μm wide, canal 5–6 μm wide in vivo (250–500 μm long, 20–25 μm wide, canal 5 μm, fixed), not widened entally, projecting into ampulla, ental bulbs about 40–55 μm wide, fixed. Ampullae are surrounded distally by about 9–12 sessile diverticula of varying size : length 24–45 μm (fixed). Sperm in a circle in lumen of ampullar distal part. Diameter of ampulla and diverticula together 110–150 μm, mostly no sperm in the diverticula. Separate openings into oesophagus dorso-laterally. 1–4 mature eggs at a time.

Figure 5. 

Micrograph of Fridericia floriformis sp. n. A Sperm funnels with very visible long spermatozoa B Sperm funnels of paratype 121.15 slide No. 2295 (marked with arrows) C Male copulatory apparati, with well-developed muscle of paratype P.212.16 slide No. 2434 latero-ventral view D Male copulatory organs, ventral view (marked with arrows) E Bursal slit F–J Spermathecae (holotype NIBRIV0000813661, slide No. 2437, I–J Spermathecal ampullae of paratype P. 121.16 slide No. 2434 where the ampullar diverticula are visible on all sides around the ampullae). A, D–G in vivo B fixed, stained C, H–J fixed, not stained. Scale bars: 50 μm.

Named after the shape of the spermathecal ampulla (more diverticula), which resembles a flower (flos, floris= flower, and formis = shaped as, Latin).

Sequences deposited in GenBank: MH128729-MH128733 (ITS), MH124586-MH124589 (CO1), MH124597-MH124598 (H3).

In South Korea, at sites 8 and 9, Jeju Island, Yongnuni-orums, clayey soil, meadows.

There are only three species (F. paraunisetosa Xie et al., 2000, F. ventrochaetosa Nagy, Dózsa-Farkas & Felföldi, 2018 and F. callosa Eisen, 1878) among all Fridericia species, which possess more diverticula of spermathecae and the lateral chaetal bundles absent or incomplete, varying with 0, 1 or maximum 2 chaetae. Fridericia paraunisetosa can easily be distinguished from F. floriformis sp. n. based on the following characters: smaller size (5.0–7.8 mm long vs. 8–17.3 mm, fixed), lateral chaetal bundles absent, ventrally only one chaeta per bundle (vs. 2–4 chaetae ventrally and 0–2 laterally), dorsal pores only from XVIII (vs. from VII), brain incised anteriorly (vs. convex), oesophageal appendages stout and unbranched (vs. with branches at the end) (Xie et al. 2000). Fridericia ventrochaetosa could be distinguished from the new species by the total absence of the lateral chaetae and having spermathecal diverticula with stalk (vs. sessile) (Nagy et al. 2018). The new species is similar to F. callosa in most traits (e.g., body size, segment number, strong body wall, thick cuticle, chaetal arrangement, number of preclitellar nephridia, position of chylus cells, the length of sperm), but the main differences between the two species are: in F. callosa the collar of sperm funnel not narrower than funnel body (Fig. 6A) (vs. narrower, Fig. 5A, B), seminal vesicle 2–3 segment large (vs. only in XI and not conspicuous). The spermathecae very variable in F. callosa (probably species complex) with or without diverticula, and the maximum number of diverticula is 6 (Eisen 1878, 1879; Christensen and Dózsa-Farkas 1999; Schmelz 2003). From the material collected in Siberia in 1994, some stained slides were prepared now. On these slides, it was visible that the few diverticula are oriented towards the proximal ampullar part (Fig. 6B–C) in contrast to the spermathecae of the new species, which always have many diverticula or diverticula-like protrusions surrounding the ampullae (Fig. 5F–J).

Figure 6. 

Micrograph of a Fridericia callosa specimen which has spermathecae with diverticula, collected from Siberia in 1994 (Christensen and Dózsa-Farkas 1999) fixed and stained on slide. A X–XII (sperm funnel marked with black arrow, male copulatory apparati marked with white arrow, lateral view) B–C Spermathecae (diverticula marked with arrows lateral view).