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Figure 1 in Enchytraeus demutatus sp. nov. (Enchytraeidae, Oligochaeta) has characters hitherto unrecorded in the genus

Figure 1. Enchytraeus demutatus sp. nov. (A) Anterior body end, lateral view, paratype ZMB 12203. Shaded-grey: pharyngeal pad, afferent fascicles and pharyngeal glands, a functional unit. (B) Anterior body end, ventral view, holotype, pharyngeal glands omitted. Shaded grey: ventral nerve cord. Brain and pharyngeal pad indicated by dotted lines. (C) Spermatheca, as seen in living specimen (comp. Fig. 2C). (D) Male reproductive organs in XI, XII, paratype ZMB 12205. Shaded grey: thickened lip on male bursa, surrounded by bursal slit. Asterisk: spherical accessory gland, attached to elongated gland. A, B, D from stained whole mounts, C from live photograph. Scale bars = 100 µm (same bar for A, B, D).

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Figure 3 in Enchytraeus demutatus sp. nov. (Enchytraeidae, Oligochaeta) has characters hitherto unrecorded in the genus

Figure 3. Phylogenetic tree based on a concatenation of six genetic markers, estimated using Bayesian inference. Posterior probabilities below 0.8 not shown. Scale bar indicates expected number of substitutions per site. For authorities of names see Table 1.

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Figure 2 in Enchytraeus demutatus sp. nov. (Enchytraeidae, Oligochaeta) has characters hitherto unrecorded in the genus

Figure 2. Enchytraeus demutatus sp. nov. (A) Pharyngeal glands and further details in III–VI, dorsal view. (B) Clitellum, ventrolateral view, specimen flattened. (C) Spermatheca. The dots in the ectal duct wall and the ectal gland are cell nuclei. (D) Coelomocytes. (E) Anterior clitellum, midventral part with bursal slits. Photographs A, E from paratype specimens, stained whole mounts, B–D from living specimens, not preserved; in all, anterior body end to the top. Scale bars 100 µm in A, B, E, 50 µm in C, D.

Fig. 12. Enchytraeus Henle, 1837 in New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Croatia

Fig. 12. Enchytraeus Henle, 1837 species tree, based on COI, H3 and 16S rRNA gene (Bayes analysis, *BEAST). Posterior probabilities greater than 0.5 are shown at the nodes. Accession codes of sequences with collection information are given in Table 1. Abbreviation: n = number of included specimens for each species. Scale bar = 0.07 substitutions per nucleotide.

Fig. 8 in New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Croatia

Fig. 8. Micrograph of Enchytraeus krumbachi (Čejka, 1913). A–B, D, F. In vivo. C, E, G–H. Fixed, stained. A–B. Sperm ducts (thick-wall portion marked with black arrow, thin-wall portions marked with white arrows). C. Large sperm sacs with more lobes (slide 2868, ELTE). D–E. Male copulatory apparates (many accessory glands marked with arrows). E. Slide 2868 (ELTE). F–H. Spermathecae (ampulla rounded without diverticula). G. Slide 2867 (ELTE). H. Slide 2872 (ELTE). Scale bars = 50 μm. Abbreviation: pbc = primer gland.

Fig. 11 in New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Croatia

Fig. 11. Maximum likelihood (ML) tree of the H3 gene for specimens of the Enchytraeus albidus species complex, based on 225 nucleotide positions using the Kimura 2-parameter substitution model. Bootstrap values greater than 50 are shown at the nodes. Accession codes of sequences with collection information are given in Table 1. Scale bar: 0.01 substitutions per nucleotide.

Fig. 10 in New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Croatia

Fig. 10. Maximum likelihood (ML) tree of the COI gene for specimens of the Enchytraeus albidus species complex, based on 568 nucleotide positions using the General Time Reversible substitution model. Bootstrap values greater than 50 are shown at the nodes. Accession codes of sequences with collection information are given in Table 1. Scale bar: 0.05 substitutions per nucleotide.

Fig. 9 in New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Croatia

Fig. 9. Micrograph of Enchytraeus irregularis Nielsen & Christensen, 1961. A–H, J, K. In vivo. I, L. Fixed, stained. I. Slide 2110 (ELTE). L. Slide 574 (ELTE). A. Brain (refractile globules marked with arrows). B. Clitellar glands, dorsolateral view. C. Coelomocytes. D. Head, anterior bifurcation of blood vessel with red blood (head pore marked with arrow). E. Chloragogen cells. F. Male copulatory organ (lateral view). G. Sperm funnels. H. Segments XII–XIII (male copulatory organ marked with black arrow, extra sexual gland marked with white arrow). I. Multiple copulatory glands (lateral view, primary gland marked with arrow). J. Extra sexual glands in XIII, ventro-lateral view. K–L. Spermathecae (diverticula marked with arrows). Scale bars: A–B, D–L = 50 μm; C = 20 μm. Abbreviation: s = sperm sacs.

Fig. 7 in New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Croatia

Fig. 7. Micrograph of Enchytraeus krumbachi (Čejka, 1913). A–B, D–F, H–I. In vivo. C, G. Fixed, stained. A. Chaetae in ventral bundles preclitellarly (surplus chaetae marked with arrow). B. Coelomocytes. C. Clitellar glands, dorsal view (slide 2868, ELTE). D. Clitellar glands, lateral view (near to male copulatory apparates more granular glands). E. Between the two male copulatory apparates (marked with arrows) and middle ventrally only granulocytes. F–G. Pharyngeal glands (in F first and secondary glands connected dorsally, in G these glands near to each other but not connected dorsally, the spermathecae marked with arrows). G. Slide 2867 (ELTE). H. Sperm sacs in X–XI. I. Sperm funnel. Scale bars = 50 μm. Abbreviation: sf = sperm funnel.

Fig. 6 in New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Croatia

Fig. 6. Micrograph of Enchytraeus albidus Henle, 1837 s. str. A–C, E–H, K–L. In vivo. D, I. Fixed, stained. J. Fixed, not stained. A. Chaetae in ventral bundles preclitellarly. B. Clitellar glands, dorsal view. C–D. Ventral recess between the lip-like folds in XII. D. Slide 3047 (ELTE). E–F Sperm ducts (In F the ciliated canal marked with arrow). G. Nephridium. H. Sperm funnel. I. Multiple male glands (larger primary bulb marked with arrows) (slide 3048, ELTE). J. Lip-like fold (slide 3028, ELTE). K–L. Spermathecae (diverticula marked with black arrow, glands at ectal duct marked with white arrow). Scale bars = 50 μm.

Fig. 5 in New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Croatia

Fig. 5. Micrograph of Enchytraeus andrasiformis sp. nov. A–C, F–G, I, K. Holotype (slide 3132, En. 4; ELTE). A–C, F–G, K. In vivo. D–E, H–J, L. Fixed, stained. A. Brain. B. Clitellar glands, dorsal view. C. Clitellar glands, lateral view (three eggs visible). D. Paratype (slide 3094, P.145.2, ELTE), chaetae in ventral bundles with surplus chaetae, preclitellarly. E. Paratype (slide 3093, P.145.1, ELTE), pharyngeal glands. F. Coelomocytes. G. Sperm funnel. H. Paratype (slide 3094, P.145.2). J. Paratype (slide 3094, P.145.2, ELTE). L. Paratype (slide 3093, P.145.1, ELTE). H–I. Multiple male glands (large primary bulb marked with black arrows, extra sexual glands marked with white arrows). J–L. Spermathecae. Scale bars = 50 μm.

Fig. 1 in New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Croatia

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.

Fig. 4 in New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Croatia

Fig. 4. Micrograph of Enchytraeus andrasi sp. nov. A–C, E, G–I, K. In vivo. D, F, J. Fixed, stained. A. Brain. B. Chaetae in ventral bundles preclitellarly (surplus chaetae marked with arrow). C. Clitellar glands of holotype, dorsal view. D. Paratype (slide 2870, P.144.1, ELTE), pharyngeal glands. E. Clitellar glands of holotype, ventral view (male lip-like folds marked with arrows, between the folds the glands absent: marked with *). F. Paratype (slide 2870, P. 144.1, ELTE), nephridium at 7/. G. Sperm duct. 8 H. Sperm funnel. I. Sperm duct and many small eggs. J. Holotype (slide 2871, En.3, ELTE), multiple male glands of holotype (large primary bulb marked with arrow). K. Spermatheca (large diverticulum marked with short black arrow, small diverticula like protrusions marked with white arrows, ectal duct covered with glands marked with long, black arrow). Scale bars = 50 μm.

Fig. 2 in New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Croatia

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.

Fig. 3 in New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Croatia

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.

FIGURE 3 in A new species of Enchytraeus (Enchytraeidae, Oligochaeta) from the profundal of Lake Van, the world's largest soda Lake (Turkey, East Anatolia)

FIGURE 3. Enchytraeus polatdemiri sp. n. Drawings from adult whole-mounted specimens, anterior end to the top (A) or to the left (C-D). A. Segments IV-VI, dorsal view, fragment of holotype, station 3. Dark grey: pharyngeal glands. B. Segments IV- VI, dorso-lateral view, fragment of paratype specimen, strongly contracted, station 3. Light-grey: dorsal blood vessel. Darkgrey: pharyngeal glands. C. Male copulatory organ, latero-dorsal view, with portions of convoluted vas deferens, Station 24. D. Same object as C, but focusing plane c. 30 µm beneath, vas deferens not shown. bu, bursa, ental tip; dv, dorsal blood vessel; ec, vas deferens, ectal portion, near male pore; eg, spermathecal ectal glands; en, vas deferens, ental portion, near sperm funnel; es, inner epidermal surface of bursa; gc, clitellum cells, granulocytes; ge, glandular thickening of epidermis around spermathecal ectal pores; lc, lateral chaetae; ml, male exterior lip, devoid of clitellar gland cells; mp, male pore; oa, oesophageal appendage; oe, oesophagus, compressed; pb, primary male bulb; pg, pharyngeal gland; rm, retractor muscles; sa, spermathecal ampulla; sp, secondary male papilla; se, spermathecal ectal duct.

FIGURE 4 in A new species of Enchytraeus (Enchytraeidae, Oligochaeta) from the profundal of Lake Van, the world's largest soda Lake (Turkey, East Anatolia)

FIGURE 4. Enchytraeus polatdemiri sp. nov. and selected specimens of other Enchytraeidae species; Bayesian trees based on: A, COI sequences; B, H3 sequences; C, the concatenated dataset. Numbers on nodes represent posterior probabilities values.

FIGURE 2 in A new species of Enchytraeus (Enchytraeidae, Oligochaeta) from the profundal of Lake Van, the world's largest soda Lake (Turkey, East Anatolia)

FIGURE 2. Enchytraeus polatdemiri sp. n. Drawings from whole-mounted specimens. A. Complete adult paratype specimen (on slide), stained whole mount, station 21. Latero-ventral view in 0-VI, ventro-lateral view in XI-XIII. B-D. Chaetae as seen in adult paratype specimens (unstained whole mounts) from station 24. B. Ventral bundle, segment VII. C. Chaeta from a posterior segment. D. Chaeta from a penultimate segment. E. Clitellum ventrally of a flattened specimen, paratype, unstained fragment, mounted, adult specimen from station 3. Light-grey: anterior and posterior border cells; medium grey: area with alternating granulocytes and hyalocytes; dark-grey: region with exclusively granulocytes. Dotted lines: Male glandular bulb and accessory glands beneath bursa. Abbreviations: br, brain; cb, clitellum, anterior border cells; lc, lateral chaetal bundle; ml, male exterior lip, extending mediad, covering bursa; mo, male copulatory organ; oo, oocyte; sf, sperm funnel; st, spermatheca; sv, seminal vesicle; vc, ventral chaetal bundle.

FIGURE 5 in A new species of Enchytraeus (Enchytraeidae, Oligochaeta) from the profundal of Lake Van, the world's largest soda Lake (Turkey, East Anatolia)

FIGURE 5. Barcode gap analysis of COI sequences performed by ABGD (Automatic Barcode Gap Discovery, Puillandre et al. 2012) (comp. Fig. 4). Histograms show the distribution of pairwise genetic distances (uncorrected p-distances) between each pair of specimens. The arrow indicates the gap that allows to distinguish intraspecific (left) and interspecific (right) distances for the COI region.