New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Croatia Author Nagy, Hajnalka 13D87FB8-C834-4CD7-AC35-219D77009297 Department of Microbiology, ELTE Eötvös Loránd University, Pázmány Péter sétány 1 / C, H- 1117 Budapest, Hungary. Hungarian Natural History Museum, Baross utca 13, H- 1088 Budapest, Hungary. Department of Systematic Zoology and Ecology, ELTE Eötvös Loránd University, Pázmány Péter sétány 1 / C, H- 1117 Budapest, Hungary. Institute of Aquatic Ecology, Centre for Ecological Research, Karolina út 29, H- 1113 Budapest, Hungary. nhajni6@gmail.com Author Dózsa-Farkas, Klára 4A08693E-3C7D-432E-8BCA-CBBDF7E36D02 kdozsafarkas@gmail.com Author Felföldi, Tamás F0CD45C8-0140-4E52-A996-080FE186EB72 Department of Microbiology, ELTE Eötvös Loránd University, Pázmány Péter sétány 1 / C, H- 1117 Budapest, Hungary. Hungarian Natural History Museum, Baross utca 13, H- 1088 Budapest, Hungary. Department of Systematic Zoology and Ecology, ELTE Eötvös Loránd University, Pázmány Péter sétány 1 / C, H- 1117 Budapest, Hungary. Institute of Aquatic Ecology, Centre for Ecological Research, Karolina út 29, H- 1113 Budapest, Hungary. tamas.felfoldi@gmail.com text European Journal of Taxonomy 2023 2023-05-26 870 107 145 http://dx.doi.org/10.5852/ejt.2023.870.2123 journal article 53501 10.5852/ejt.2023.870.2123 bac760e2-c211-419a-a1ca-5f5af426268f 2118-9773 7986633 508560B7-BD89-472A-B3FD-F48851B024C3 Enchytraeus adrianensis sp. nov. urn:lsid:zoobank.org:act: B49B8279-3D90-408F-B45C-BF89ACFC3C2B Figs 1A , 2–3 , Tables 1–2 Diagnosis (1) Body length 14–30 mm (in vivo), segment number 51–84; (2) chaetae maximum 4 per bundle, straight with ental hook; (3) clitellum in XII–XIII, hyalocytes and granulocytes in dense transverse rows dorsally and laterally (or in a reticulate pattern), mostly only granulocytes between the male pores; (4) epidermal gland cells in 3–4 rows/segments, often brownish; (5) four pairs of nephridia preclitellarly; (6) pharyngeal glands widely or slightly connected dorsally and with ventral lobes; (7) dorsal blood vessel origin from XIV–XV, blood colourless; (8) sperm funnel cylindrical, 350–690 μm long, 2–4 × as long as wide in vivo; (9) vasa deferentia tripartite, extending into XV–XIX, the slender part ciliated, the thickened middle section not ciliated; (10) male glands multiple: one large rounded primary bulb (60– 125 μm long and 50–95 μm wide), and 6–9 smaller secondary glands, ventral recess between the male copulatory organs absent; (11) spermathecal ectal duct (90–160 μm long in vivo) covered with gland cells, ampulla oval or spherical (70–115 μm wide) with wide wall and without diverticula, connecting with oesophagus; (12) 2–7 mature eggs present at a time. Etymology Named after the Adriatic Sea where it was found. Material examined Holotype CROATIA • Istria , Kamenjak Peninsula , Adriatic Sea, Kale Cove, seashore, decaying Zostera debris ; 44°51′13.0″ N , 13°58′50.5″ E ; 3 Apr. 2019 ; Júlia Török leg., from culture: 15.10.2019 , En.2. slide 2842 ; ELTE . Paratypes (21 specs) CROATIA • 1 spec. ; same collection data as for holotype; slide 2841a,b ; P.143.1; ELTE • 1 spec. , last 19 segments, 3 mm used for DNA analysis (No. 1265, ID number); same collection data as for holotype; slide 2717 ; P.143.2; ELTE • 1 spec. ; same collection data as for holotype; slide 2748 ; P.143.3; ELTE • 1 spec. , last 18 segments, 2.3 mm used for DNA analysis (No. 1324, ID number); same collection data as for holotype; slide 2750 ; P.143.4; ELTE • 1 spec. , last 15 segments, 3 mm used for DNA analysis (No. 1266, ID number); same collection data as for holotype; slide 2751 ; P.143.5; ELTE • 1 spec. ; same collection data as for holotype; 2760 ; P.143.6; ELTE • 1 spec. ; same collection data as for holotype; slide 2822a–c ; P.143.7; ELTE • 1 spec. ; same collection data as for holotype; slide 2827a,b ; P.143.8; ELTE • 1 spec. ; same collection data as for holotype; slide 2850 ; P.143.9; ELTE • 1 spec. ; same collection data as for holotype; slide 2851a,b ; P.143.10; ELTE • 1 spec. ; same collection data as for holotype; slide 2852a,b ; P.143.11; ELTE • 1 spec. ; same collection data as for holotype; slide 2853 ; P.143.12; ELTE • 1 spec. [last 16 segments, 2.2 mm used for DNA analysis ( No. 1379 , ID number)]; same collection data as for holotype; slide 2854 ; P.143.13; ELTE • 1 spec. ; same collection data as for holotype; slide 2855 ; P.143.14; ELTE • 1 spec. , last 22 segments, 2.7 mm used for DNA analysis ( No. 1380 , ID number); same collection data as for holotype; slide 2856 ; P.143.15; ELTE • 1 spec. ; same collection data as for holotype; slide 2857 ; P.143.16; ELTE • 1 spec. ; same collection data as for holotype; slide 2858 ; P.143.17; ELTE • 1 spec. ; same collection data as for holotype; slide 2859 ; P.143.18; ELTE • 1 spec. ; same collection data as for holotype; slide 2861 ; P.143.19; ELTE • 1 spec. ; same collection data as for holotype; slide 3010 ; P.143.20; ELTE • 1 spec. ; same collection data as for holotype; slide 3011 ; P.143.21; ELTE . Fig. 1. Schematic drawing of spermathecae of the specimens studied. A . Enchytraeus adrianensis sp. nov. B . E. krumbachi ( Čejka, 1913 ) . C–D . E. andrasi sp. nov. E . E. andrasiformis sp. nov. F . E. albidus Henle, 1837 . G . E. irregularis Nielsen & Christensen, 1961 . Other material CROATIA • 15 specs ; same collection data as for holotype; ELTE • 3 specs (only for DNA analysis); same collection data as for holotype; ELTE . Description MEASUREMENTS . Large specimens. Holotype 22.8 mm long, 570 μm wide at VIII and 770 μm at clitellum, fixed, segment number 60. Body length of paratypes 14–30 mm , width 450–780 μm at VIII and 550– 1000 μm at clitellum, in vivo. Length of fixed specimens 7.7–24.9 mm (in one specimen 28.2 mm , width 410–730 μm at VIII and 490–950 μm at clitellum). Length of the first 12 segments 2.2–4.8 mm , after fixation. Segment number 51–84 (N = 34). CHAETAE . Chaetal formula according to Schmelz & Collado (2010) : 2,3–2,3:3,4–2,3. Chaetae straight with ental hook, about equal in size within bundle, 72–112μm × 5–7.5 μm preclitellarly and 70–100 μm × 5–7.5 μm posteriorly. In ventral preclitellar bundles of some specimens 3 chaetae, and only one or two bundles with 4 chaetae, in other specimens 4 chaetae in most of all other bundles. Often 2–3 surplus chaetae near the bundles ( Fig. 2B ). Chaetae in XII absent ventrally, but 2–3 per bundle present laterally. HEAD PORE . At 0/I. EPIDERMAL GLANDS . Often brown, arranged in 3–4 rows per segment. CLITELLUM . Girdle-shaped, in XII–XIII, hyalocytes and granulocytes in dense transverse rows dorsally and laterally ( Fig. 2C ), but in well-developed sexual condition gland cells in reticulate pattern ( Fig. 2D ). Mostly only granulocytes between male pores ( Fig. 2E–F ). BRAIN . About 1.3–1.8 × as long as wide, rounded posteriorly, sides slightly merging anteriad often with 2 small aggregations of refractile globules ( Fig. 2A ). OESOPHAGEAL APPENDAGES . Pair of blind-ending tubes in III / IV, with common root inserting dorsally behind pharyngeal pad. Mostly all primary pharyngeal glands widely or slightly connected dorsally with ventral lobes ( Fig. 2H ). In some specimens first pair separate ( Fig. 2G ). Ventral lobes of third pair largest. DORSAL BLOOD VESSEL . From XIV–XV, blood colourless. Anterior bifurcation near prostomium. NEPHRIDIA . Four pairs of preclitellar nephridia from 6/ 7–9 /10, anteseptale funnel only, postseptale bulged, efferent duct short, origin postero-ventrally. COELOMOCYTES . Oval or narrowed at one end, texture granulated, about 24–40 μm long, in vivo ( Fig. 2I– J ) [15–30 μm, fixed ( Fig. 2K )]. In addition, many shining, hyaline, round or tetragonal corpuscles [diameter (10–19 μm)] also present ( Fig. 2J ), which at lower magnification shine like grains of sand. In young specimens corpuscles always fewer. Note, corpuscles not visible after fixation. SUBNEURAL GLANDS . Absent. Fig. 2. Micrograph of Enchytraeus adrianensis sp. nov. A–D, I–J . In vivo. E–H, K–L . Fixed, stained. A . Brain (small aggregations of refractile globules marked with arrows). B . Chaetae in a ventral bundles with 3 surplus chaetae. C . Clitellar glands in regular rows. D . Clitellar glands in reticulate pattern. E . Paratype (slide 2748, P.143.3, ELTE). F . Slide 2684 (ELTE). H . Slide 2761 (ELTE). K . Paratype (slide 2851, P.143.10, ELTE). L . Paratype (slide 2760, P.143.6). E–F . Clitellar glands, ventral view (male copulatory organs marked with black arrow, between these organs the granulocytes marked with white arrows). G . Paratype (slide 2856, P.143.15, ELTE), first pharyngeal glands free dorsally. H . All pharyngeal glands connected dorsally (spermathecae marked with arrows). I–K . Coelomocytes (in J the small hyaline, refracting corpuscules also present). L . Large paired lobes of sperm sacs. Scale bars: A–I, K–L = 50 μm; J = 20 μm. SPERM SACS . Two or three paired lobes of sperm sacs, very large, filling the coelom of IX /X–XI ( Fig. 2L ). Testes and sperm funnels in XI, ovaries, male pores and glands in XII. SPERM FUNNELS . 350–690 μm long in vivo (300–500 μm, fixed), 1.7–4 × as long as wide, collar narrower than funnel body ( Fig. 3A ). Vasa deferentia distinctly tripartite, long, extending into segments XV–XIX. After sperm funnel and before male opening, vasa deferentia slender and ciliated (20–40 μm wide in vivo and fixed equally), middle part thickened (50–95 μm wide) and unciliated ( Fig. 3B ). SPERMATOZOA . 75–100 μm long, heads 20–30 μm, fixed. MALE COPULATORY ORGANS . Male glands multiple: one large rounded primary bulb near male pore, and 6–9 smaller secondary glands, mostly 8 (usually 4–5 on each side, but sometimes 5 in one side and only 3 at other). Besides, near male opening in middle 2–3 smaller additional glands. Glands arranged roughly in semicircle around male pore and primary bulb (diameter 200–350 μm, fixed) ( Fig. 3C–F ). Primary bulb 60–125 μm long and 50–95 μm wide, secondary glands near to primary glands longer than outside ones (40–100 μm long and 20–50 μm wide). Male pores covered by lip-like folds (100–160 μm wide), ventral recess absent (all data in fixed specimens). SPERMATHECAE ( Figs 1A , 3G–I ). With ectal duct, ampulla without diverticula, separate openings into oesophagus. Spermathecal ectal pore at 4 / 5 . Ectal duct with different length (90–160 μm in vivo, 70– 160 μm, fixed), covered with gland cells (70–117 μm widely in vivo and 60–100 μm widely, fixed). In most specimens, ectal duct near ampulla without glands for short stretch, length about 7–12 μm. Canal of ectal duct 6–9 μm wide, widening entad. Ampulla oval or spherical, about 70–115 μm wide and 75–150 μm long, with distinct, about 13–20 μm wide walls, lumen with masses of spermatozoa, ental duct very short, connecting laterally with oesophagus. 2–7 mature eggs present at a time. Differential diagnosis In the Enchytraeus albidus species complex as circumscribed in Erséus et al. (2019) , the new species and two other species of which sequences are available ( E. albellus , E. cf. krumbachi ) have tripartite vasa deferentia. In E. albellus , the ductus is ciliated in its full length, whereas in the new species and in E. krumbachi the thickened middle part is not ciliated. Moreover, E. albellus is different, because its spermathecal ampulla is irregular sac-like with one smaller diverticulum dorsally, but in the new species the ampulla is oval or spherical with a well developed wall without diverticula. The new species is closely related to E. krumbachi based on morphological and molecular data. Morphological similarity is primarily observable in the form of the spermatheca and the absence of the ventral recess of the clitellum. In both species the ampullae are without diverticula, but in E. krumbachi round and the diameter is slightly larger (70–140 μm in the Ligurian Sea specimens of E. krumbachi collected in this study but in the new species often oval and only 70–115 μm). Furthermore, the new species is bigger (segment number 51–84, body length 7.7–24.9 mm and 490–950 μm wide at clitellum vs segment number 40–66, body length 7–14 mm and 500–860 μm wide at clitellum, in vivo). No doubt, that the ventral recess absent in both species, but in the male copulatory organ the primary bulb about the same size in both species. In the new species mostly 8 long, narrower secondary glands are in a semicircle, around male pore and primary bulb, but in E. krumbachi the primary bulb is surrounded with very much smaller secondary glands. Although the molecular data also showed a close relationship between the two species, they still supported a certain species-level separation. Enchytraeus polatdemiri differs from the new species by its smaller size (segment number 40–55, length 7.5–11 mm and 410– 580 μm wide at clitellum), and the sperm funnel is 5 × as long as wide (vs 1.7–4 × as long as in the new species) and the vasa deferentia are not tripartite, ciliated throughout, and mostly confined to XII (vs XV–XIX). Enchytraeus moebii is similar to the new species in the form of the male apparatus (large primary bulb and smaller accessory glands especially on the basis of figure of Michaelsen (1886 : fig. 3 pl. II), but the principal differences are that the vasa deferentia are not tripartite, ciliated throughout and the collar of sperm funnel is wider than the funnel body (vs collar narrower than funnel body in the new species). Enchytraeus irregularis differs principally from the new species by the well developed diverticulum of the spermatheca, by the smaller primary bulb of male apparatus (diameter 40–80 μm vs 60–125 μm in the new species), and the vasa deferentia of E. irregularis are uniform, ciliated throughout and confined to XII. Very characteristic trait for E. irregularis is the oval or squarish glandular structure without external orifice (‘accessory sexual glands’) after or before male copulatory organs in XIII, XII or XI but in some specimens these organs may be absent. Fig. 3. Micrograph of Enchytraeus adrianensis sp. nov. A, C, G–H . In vivo. B, D–F, I . Fixed, stained. A . Sperm funnels. B . Paratype (slide 2748, P. 143.3, ELTE), sperm ducts (slender part marked with white arrow, wider part with black arrows). C–F . Multiple male glands (E–F primary bulb marked with arrow). D . Paratype (slide 2748, P.143.3, ELTE). E . Paratype (slide 2748, P.143.3, ELTE). F . Holotype (En.2. slide 2842, ELTE). I . Paratype (slide 2827, P. 143.8, ELTE). F . Holotype (En.2. slide 2842, ELTE). G–I . Spermathecae (ectal duct covered with glands marked with white arrows, ampullae filled with sperm in it marked with black arrows).Scale bars = 50 μm. For similarities and differences of the species of Enchytraeus studied by us, see Table 2 . Distribution and habitat Kale Cove, Adriatic Sea, Kamenjak Peninsula, Istria , Croatia , decaying Zostera debris.