Two new enchytraeid species from Jeju Island, Korea (Annelida, Clitellata) Author Dozsa-Farkas, Klara Author Felfoeldi, Tamas Author Nagy, Hajnalka Author Hong, Yong text ZooKeys 2019 824 87 108 http://dx.doi.org/10.3897/zookeys.824.25544 journal article http://dx.doi.org/10.3897/zookeys.824.25544 1313-2970-824-87 549FF67544A742FEADF974E318EE0AA5 549FF67544A742FEADF974E318EE0AA5 Achaeta multisacculata sp. n. Figs 1 A-D , 2, 3 Type locality. Clayey soil, meadow (site 9), Yongnuni-orum, Gujwa-eup, Jeju Island, South Korea. Holotype. 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. Diagnosis. 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. Description. 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 clitel lum 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, 3 H-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, 3 K-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. Etymology. Named after the high number of 'pyriform glands' (sacculus = saccule, Latin). Molecular data. Sequences deposited in GenBank: MH128727-MH128728 (ITS), MH124584-MH124585 (CO1). Distribution. In South Korea, at sites 8-9, Jeju Island, Yongnuni-orums, clayey soil, meadows. Morphologically similar species. 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).