Fridericia ciliotheca, Schmelz, Rüdiger M. & Collado, Rut, 2013

Schmelz, Rüdiger M. & Collado, Rut, 2013, Enchytraeidae (Oligochaeta, Annelida) from a field site in Portugal, with the description of five new species and a redescription of Enchylea heteroducta Nielsen & Christensen, 1963, Zootaxa 3647 (2), pp. 307-328 : 316-322

Fridericia ciliotheca sp. nov.

( Figs 4 A–D, 5C, Table 2 )

Holotype. MNHML MB29-000316, adult spcm, stained whole mount. Portugal, Coimbra, in soil from the experimental field area of the Coimbra Higher School of Agriculture (ESAC), meadow site ( Table 2 ); II 2012.

Paratypes. 13 spms. MNHML MB29-000317, –318, stained whole mounts, 1 adult, 1 subadult. ZMH OL 14526, 5 adults, unstained whole mounts. ZMH OL 14527, fixed in Bouin's fluid, preserved in 70% ethanol: 9 spms. ZMH OL 14528, fixed in 70% ethanol, preserved in 100% ethanol: 2 spms.

Other material. 11 spms investigated in vivo, preserved in collective sample vials in the authors' collection.

Etymology. Named for the ciliar movement in the spermathecal ampulla.

Diagnosis. More than 50 segments, max. 4 chaetae per bundle, clitellum saddle-shaped, coelomo-mucocytes without refractile vesicles, nephridia present at 10/11, chylus cells post-clitellar, no seminal vesicle, sperm heads c. 120 μm, sperm funnel small, spermathecae separate entally, no ectal gland, two stalked diverticula with ciliated subchamber, ampulla constricted ectally, with ciliar movement near ental bulb.

Description. Worms with transparent and opaque parts and often greenish gut (viv), slow body movements. Length 13–15 mm (viv), 10–12 mm (fix), diameter 0.30–0.35 mm at XII–XIII, 0.25–0.28 mm in rest of body (viv, fix). Segment number 55–59 in fully adult worms (N=10), often less in subadults. Chaetae max. 4 per bundle; formula 4 – 4,3,2: 4 – 4,3,2. Mostly 4 chaetae per bundle in anterior body half (e.g. down to XXVII in a specimen with 58 segments) and 2 chaetae per bundle in posterior body half, 3 chaetae only in bundles of a few mid-body segments. Anterior chaetae max. 65 μm long and c. 5 μm wide, inner chaetae c. 3/5 the size of the outer, laterals slightly smaller (e.g. 56 μm) than ventrals; postclitellar chaetae not reduced in size, chaetae in caudal segments not longer than outer chaetae in anterior segments, but slightly thicker (up to 6 μm). Single packages of detached chaetae occasionally present in body cavity. Epidermal gland cells pale, quasi-rectangular, in 3 rows in anterior segments, most cells in mid-row at chaetal level, mid-dorsally and mid-ventrally no cells; in some specimens cells inconspicuous or apparently absent. Body wall 20–30 μm thick, cuticle thin (<1 μm), preclitellar septa not conspicuously thickened.

Brain egg-shaped (viv), c. 100: 70 μm (fix), rounded on all sides, anteriorly with very slight convexity, almost truncate. Pharyngeal glands dorsally united in IV, V, separate in VI; dorsal and ventral lobes increasing in size from IV to VI; in IV ventral lobes separate from dorsal lobes. Oesophageal appendages elongate with few terminal branches, extending to VI. Chloragocytes with greenish or greyish vesicles. Chylus cells in XV–XIX, occupying 3.5–4 segment lengths, canals not widened into lacunae. Dorsal blood vessel from XXIII–XXIV. Midgut pars tumida not seen. In two consecutive posterior segments, intestinal epithelium with a network of fine canals. Preclitellar nephridia 5 pairs, from 6/7 to 10/11, constricted at septum, length ratio anteseptale: postseptale between 3: 4 and 2: 3, adseptal to medial rise of efferent duct, no terminal vesicle. Postclitellar nephridia from 13/ 14, constriction less marked, also with large anteseptale and subterminal rise of efferent duct, no terminal vesicle. Coelomo-mucocytes small, length 20–30 μm (fix), pale, 1.5– 2 x as long as wide, matrix with strongly blurred vesicles, almost hyaline, nucleus distinct, with 1 or 2 minute nucleoli; margins of cells in oblique position appear dark (light refraction); coelomo-lenticytes 7–8 μm long. Both cell types numerous but not filling body cavity. Cell aggregations with grey tint, cells appear to stick to one another.

Clitellum saddle-shaped with cells in c. 35 dense, transverse rows, conspicuous but not much elevated, cell height c. 15 μm, 20 μm in contracted worms (fix), granulocytes and hyalocytes alternating, diameters 8 and 15 μm, respectively (fix); hyalocytes extending down to ventro-lateral margins; female pores surrounded by granulocytes only. Mid-ventral aclitellar field wide at level of bursal slits, extending laterally beyond slits; in the rest, aclitellar field narrower than distance between ventral chaetal bundles, narrowest at level of female pores. Seminal vesicle absent, developing sperm in anterior half of XI. Spermatozoa> 300 μm long, heads c. 120 μm. Sperm funnel conical/cylindrical, variable in shape and often of irregular outline, about half as long as body diameter, collar narrower than funnel body, with few spermatozoa attached. Vas deferens 20 μm wide (fix) proximally near sperm funnel, continuously tapering to 9 μm distally (fix) near male gland. Male copulatory organ: male gland distinctly longer than wide, e.g. 120 μm long, 70 μm wide, 50 μm high (fix), bursal slit H-shaped. Subneural glands and other accessory glands absent. Spermatheca: no ectal gland; ectal duct about as long as body diameter, c. 18 μm wide (viv), not widened in distal part; ental bulb not projecting into ampullar lumen, only slightly wider than ectal duct, ectal duct canal straight throughout; ampullae with widely separate attachment to oesophagus dorso-laterally; each ampulla 1.2–1.5x as long as wide, rounded entally and constricted ectally at transition to diverticula; walls 4–6 μm thick, uniform, no histological differences observed, with smooth outer and inner surface, inner surface somewhat uneven, occasionally wavy, as a transitory state during contraction. Diverticula inserting on opposite sides of ental bulb at base of ampulla and projecting obliquely ectad, distance (= maximum width of spermatheca) c. 60–70 μm, c. 1/4 body diameter. Each diverticulum c. 1.4x as long as wide, diameter c. 20 μm, lumen subdivided into peripheral sperm-containing chamber and ciliated sub-chamber; both chambers connected by a porus; peripheral chamber wider than long, with dense sperm roll circulating; sub-chamber longer than wide, widely connected with ampullar lumen. Ciliar movement in peripheral chamber of diverticula and also in distal part of ampulla, in vicinity of ental bulb surface ( Fig. 4 B, arrow).

Remarks. F. ciliotheca belongs to the group of Fridericia species with spermathecal diverticula that have a ciliated subchamber. The movements of the cilia keep the packages of allosperm in constant rotation, probably a means to maintain them in viable state. Additionally, ciliar movement was also observed inside the ampulla, in immediate vicinity of the ental bulb ( Fig. 4 B, arrow). The species owes its name to this peculiarity, observed here for the first time in Fridericia (and perhaps Oligochaeta in general, since to our knowledge Fridericia is the only oligochaete taxon where ciliated spermathecae occur). The character is only seen in living specimens. Insertion and length of the cilia could not be ascertained, but a continuity with the cilia in the diverticular subchamber was not evident, either. It seems, therefore, that the cilia insert in the ampullar epithelium.

Among Fridericia species with ciliated subchamber, most similar to F. ciliotheca sp. nov. is F. u l r i k a e Rota & Healy, 1999, with a maximum of 4 chaetae per bundle, saddle-shaped clitellum, long spermatozoa, elongate male glands, and separate spermathecae. Apart from the absence of cilia in the ampulla, F. u l r i k a e differs from F.

ciliotheca in the following characters: 4 pairs of preclitellar nephridia (vs. 5), seminal vesicle always large, extending over two segment lengths (vs. small, confined to XI), pharyngeal glands in VI connected dorsally (vs. not connected), spermathecae distinctly larger, with diverticula 40–50 μm wide (Rota & Healy 1999, Schmelz pers. obs.) (vs. 20 μm), ectal duct distinctly shorter than body diameter (vs. as long as body diameter). Figure 5 and Table 2 give a comparison of F. ciliotheca with the other three new species of Fridericia from the same site.

Achaeta coimbrensis sp. nov. ( Fig. 6 A–E)

Holotype. MNHML MB29-0 0 0 319, adult spcm, stained whole mount. Portugal, Coimbra, in soil from the experimental field area of the Coimbra Higher School of Agriculture (ESAC), crop site ( Table 2 ); IV 2010.

Paratypes. MNHML MB29-000320 –321, two spms, adult, subadult, stained whole mounts, same data as for holotype.

Other material. No further material available.

Etymology. Named after the city of Coimbra, Portugal.

Diagnosis. Length c. 2 mm, 18–21 segments, no pyriform or lentiform glands, oesophageal appendages in V only, no secondary pharyngeal gland lobes, dorsal blood vessel from 1/ 2 VII, abrupt widening of intestine at 1/ 2 VII, two pairs preclitellar nephridia at 6/7, 7/8, gonadal region shifted one segment forward, clitellum with narrow dorsal and wide ventral interruption, hyalocytes on dorsal body half only, in irregular, not baguette-shaped longitudinal rows, no seminal vesicle, sperm funnel and male copulatory organs small, male gland tripartite, with two small accessory glands lining larger central gland, spermathecae short, ectal pores lateral.

Description. Body length c. 2 mm (holotype 2.16 mm), diameter c. 0.1 mm (holotype: 0.085 mm at V, 0.11 mm at XI–XX) (fix). Segment number 18 (holotype), 20 (subadult paratype), 21 (adult paratype). Head pore on prostomium, a longitudinal slit. Body wall very thin, thickness 2–3 μm, layers (cuticle, epidermis, ring muscles, longitudinal muscles) barely distinguishable. No pyriform glands. No lentiform glands seen. Prostomium wider than long, with thickened frontal tissue protruding into lumen, reducing free coelomic space inside prostomium, no frontal recess observed (viv, fix); at least two pairs of papillae distinguishable.

Brain broadly pear-shaped, length 50 μm, width 30 μm, with a minute pair of prostomial ganglia. Ventral nerve cord ganglionate from V on, II–IV fused into suboesophageal ganglion. Ganglia at mid-length with large nonstaining cells ventrally ( Fig. 6 B). Two pairs of post-pharyngeal ganglia; the outer pair elongate, on afferent fascicles of pharyngeal glands. Septa 4/5–6/7 slightly thickened. Pharyngeal glands in IV–VI, all with ventral lobes, dorsal lobes united in IV and V, in VI united or separate, ventral lobes may be separate from dorsal lobes here. Secondary pharyngeal gland lobes absent. Oesophageal appendages in anterior half of V, a pair of spherical dorso-lateral bodies, lobe diameter almost equals that of dorsal pharyngeal gland lobes. Preclitellar nephridia at 6/7 and 7/8; anteseptale as long as wide, postseptale c. 2– 3 x as long as anteseptale, gradually narrowed into short efferent duct; no terminal vesicle. Postclitellar nephridia not more than 1–3 altogether, at 15/16 or 16/17 only, paired or unpaired, shape as preclitellar. Chloragocytes in one specimen with large yellow-brown vesicles, here over the entire body; coverage of intestine incomplete; first cells in IV; many chloragocytes floating freely in the coelom. Dorsal blood vessel from 1/ 2 VII. Gut abruptly widening in 1/ 2 VII, immediately behind dorsal blood vessel origin, with small inner recess of intestinal epithelium directed anteriad. Pars tumida of midgut in XII–XIV or XIII–XV, cell height more than half the gut diameter ( Fig. 6 B). Two types of coelomocytes: type I c. 25 µm long, flat, pale, inconspicuous, slightly longer than wide, with pointed projections at narrow ends; type II c. 15 µm long, filled with distinct pale vesicles. As a third type of cells in the coelom, detached chloragocytes. Coelomocytes pale and inconspicuous, chloragocytes very conspicuous (viv).

Testis and sperm funnels in X, male efferent apparatus and ovary in XI. Clitellum in XI–1 / 2XII, not developed mid-dorsally and mid-ventrally, mid-dorsal gap narrow, mid-ventral gap as wide as distance between male pores. Glands in 16–18 transverse rows; hyalocytes in four longitudinal rows, one dorsal and one dorso-lateral on each side; dorsolateral rows somewhat irregular in posterior half ( Fig. 6 C); rows not compacted, hyalocytes not protruding into coelom beneath body muscles. Testis unpaired mid-ventrally. Sperm developing freely in X, no seminal vesicle. Sperm heads c. 14 µm long (viv), total sperm length not measured. Sperm funnel comparatively small, about 1/4 as long as body diameter or less, about 2x as long as wide, barrel-shaped, oval in diameter, so length-width ratio larger in side view (1.5: 1); collar narrower than funnel body. Vas deferens 3–4 μm wide, thinnest in mid-section, widening ectally near male pore, irregularly coiled entally near sperm funnel. Male pores in XI, widely paired, on body surface, at mid-length of clitellum; male glands multiple, arranged longitudinally: one larger compact horse-shoe shaped gland surrounding male pore, 30: 25 μm, and 1–2 small accessory glands attached anteriorly and posteriorly, diameter <10 μm. Spermathecae with lateral ectal pores, blind-ending, confined to V, shorter than body diameter, projecting entad as a stiff finger or attached to inner body wall, length c. 60 μm, diameter c. 8–10 μm, ental end club-shaped, diameter 15 μm, sperm at about 2/5 length ectally, ectal duct canal lined with cuticle for only 8 μm. One mature oocyte at a time, filling out one segment.

Remarks. The new species belongs to a group of Achaeta species without pyriform glands and with lateral spermathecal pores. Within this group, a forward shift of the sexual organs (except the spermathecae) by one segment is characteristic of the European species A. pannonica Graefe, 1989 , A. diddeni Graefe, 2007 , and the South American A. hanagarthi Schmelz, 2008 . Among these three species, A. diddeni is most similar to the new species. Both have in common: small body size, low segment number (22–24 in A. diddeni ), oesophageal appendages in V only, dorsal blood vessel from VII, two pairs preclitellar nephridia at 6/7, 7/8, clitellum open dorsally and ventrally, no 'baguette'-shaped concentration of hyalocytes dorso-laterally, male reproductive system and spermathecae small. Differences between both species are as follows: A. diddeni has (1) six segmental epidermal lentiform gland cells (vs. none seen), (2) secondary (post-septal) pharyngeal glands in V and VI (vs. none), (3) the intestine widens gradually (vs. abruptly at 1/ 2 VII), and (4) accessory male glands are absent (vs. present). A. pannonica has the same pattern of similarities and differences, except for the nephridia (3 preclitellar pairs, vs. 2) and lentiform glands (more than 6 per segment, vs. none seen). The South American A. hanagarthi is also without secondary pharyngeal gland lobes but differs in segment number (> 30), preclitellar nephridia (1 pair, at 7/8), and spermathecae (long, extending into VI or VII), among other characters. Furthermore, A. coimbrensis sp. nov. appears to be smaller than the three species, but dimensions of the new species are from fixed material.

Lentiform epidermal gland cells were not found after intensive observation of living and preserved specimens, but their absence cannot be stated with certainty because these glands are sometimes difficult to see.—The variability of the pharyngeal gland lobe pattern in VI may be artifactual in parts (the adult paratype specimen is damaged in this region) and cannot be assessed here taxonomically.—The non-staining cells inside the ventral nerve cord are common in Achaeta species, but their function is still unclear. They may be neurosecretory gland cells.—The low number of type specimens is unsatisfactory, but we hope to add more material in future surveys at the type locality.

Figures

FIGURE 4. Fridericia ciliotheca sp. nov. A. Segments IV – VII, dorsal view. B. Spermathecae. Arrow: cilia in ampulla. C. Epidermal gland cells in vivo, preclitellar segments. D. Sperm funnels. A, D fix; B, C viv.

FIGURE 5. Comparison of the ventral sides of the clitellum in the four new species of Fridericia, schemati c. A. F. s o u s a i sp. nov. B. F. roembkei sp. nov. C. F. ciliotheca sp. nov. D. F. m a rg i n a t a sp. nov. Areas in dark grey: granulocytes only. Areas in light grey: granulocytes and hyalocytes alternating.

FIGURE 6. Achaeta coimbrensis sp. nov. A. Anterior body region, dorsal view. B. Segments XV, XVI, lateral view, showing intestine with pars tumida (arrow) and chloragocytes on top, nephridium, and ventral nerve cord with non-staining cells inside. Body cavity shaded grey. C. Clitellum and male reproductive system, lateral view. Shaded grey: clitellar region with only granulocytes. D. Segments IV – VII, dorso-lateral view. E. Male efferent apparatus. A – E fix. Same scale bar for A – D.