Trichodrilus tenuis Hrabě, 1960

Rodríguez, Pilar & Fend, Steven, 2025, Contributions to the knowledge of the genus Trichodrilus Claparède (Lumbriculidae, Clitellata) with the description of new groundwater species from Spain, Zootaxa 5711 (1), pp. 57-78 : 64-68

Trichodrilus tenuis Hrabě, 1960

( Figures 4 , 5 ; Table 3)

Trichodrilus tenuis Hrabě, 1960: 271 , figs. 26–28. Wachs 1967: 234; Juget & Dumnicka 1986: 240; Rodriguez & Giani 1994: 36, table 2; Juget & des Chatelliers 2001: 23, fig. 4; Achurra & Rodriguez 2008: 165, table 1; Achurra et al. 2015: 159, table 1; Des Chatelliers et al. 2009: 685; Giani et al. 2011: 91, table 1; Rodriguez-Noriega 2013: 166, table 5.3.6.1.c; Dumnicka 2014: table 2; Camacho & Puch 2021: 208.

Trichodrilus cf. tenuis Hrabě : Martin et al. 2015: 564 .

Material from Spain examined (see Table 1 for locality data) (first author’s collection):

—Argatxa spring, Bizkaia ( 18 December 1984, P. Rodriguez leg.) 1 mated, dissected individual.

—Goikoetxe Cave, Bizkaia ( 31 January, 2016, P. Rodriguez leg.) 1 mated, whole-mounted individual.

— Escalón Cave , Cantabria ( 18 September, 2008, A. Achurra leg.) 1 partly mature, dissected individual .

— La Cullalvera Cave , Cantabria ( 28 March 1988, A. Camacho leg.) 1 mated, dissected individual .

— Source of the Bustablado River , Cantabria, ( 14 March, 2003, Ana Camacho leg., donated by E. Martinez-Ansemil) 2 mated, dissected individuals .

— El Molino Spring, Bustablado , Cantabria ( 14 March, 2003, Ana Camacho leg., donated by E. Martinez-Ansemil) 1 mated, whole-mounted individual .

— La Coventosa Cave , Cantabria ( 1 April, 2003, Ana Camacho leg., donated by E. Martinez-Ansemil) 2 mated, whole mounted individuals .

—La Cubilla Cave ( 15 March, 2003, Ana Camacho leg., donated by E. Martinez-Ansemil) 1 whole-mounted individual with eggs.

— Rio Chico Cave, source of the Gandara River , Cantabria ( 5 June, 2003, Ana Camacho leg., donated by E. Martinez-Ansemil) 2 mated, whole-mounted individuals .

—Los Santos Cave ( 4 June 2003, Ana Camacho leg., donated by E. Martinez-Ansemil) 1 unmated, whole-mounted individual.

—Ojo Guareña Cave, Burgos (Pilar Rodriguez and Ana Camacho leg.) 5 dissected individuals, four of them mated and one with eggs, sampled in 18 March 1995, 3 November 1995, 30 August 2002, 22 November 2002, 24 April 2004.

— Well in Velamazan , Soria ( 13 September 1976, R. Rouch leg.) 1 whole-mounted and 2 dissected, mated individuals .

— Ginel R. source, La Magdalena Spring, Zaragoza ( 5 September 2024, Pilar Rodriguez leg.) 3 whole-mounted and 2 dissected, mated individuals .

—Well in Los Picos, Alicante ( 5 July 1986, A. Camacho leg.) 1 mated, dissected individual.

—Well in Zafra-Rio Bodión, Badajoz ( 3 November, 1988, A. Camacho leg.) 1 mated, whole-mounted individual.

—Well in Ermita San Isidro, Gibraleón, Huelva ( 4 November, 1988, A. Camacho leg.) 1 mated, dissected individual.

Description (measurements from Spanish mated individuals; those data marked with (a) are from a very small individual in Rio Chico Cave).

Number of segments 44 [Velamazan] and 60 [Ginel R. source] of the only two individuals found complete. Body diameter in X (0.14 (a)) 0.20–0.35 mm. Prostomium 84–150 µm long. Anterior secondary annulus beginning in segment III or IV (rarely in V) and extending throughout the body. Chaetae with nodulus slightly distal; in preclitellar segments chaetae 41–103 µm long in dorsal bundles, 61–107 µm long in ventral bundles, shorter in II, and dorsals about 0.7–0.9 times the length of ventrals of their respective segments; chaetae become shorter in posterior segments (to 80–90 µm) (measurements taken from two individuals).

All genital pores in the line of ventral chaetae. One pair of male pores in the posterior part of segment X, close to the septum 10/11, open on short, round to conical porophores (c. 25 µm high) ( Fig. 4A–C ). Two pairs of simple spermathecal pores in segments XI and XII, which open in the line of ventral chaetae, behind the ventral bundles. One pair of female pores, in 11/12.

Clitellum from X to XII (sometimes also in the anterior part of XIII, or extending through XIII). The pharyngeal glands present from IV or V to VII or the anterior part of segment VIII. First nephridium seen in 6/7, although fixation and orientation in whole mounts or damage during dissection makes it difficult to see this character in some individuals.

Paired testes in IX and X, one pair of ovaries in XI. Sperm sacs extend to XI– XIII; egg sacs to XII–XV. Male funnels on 9/10 and 10/11. Free portion of both anterior and posterior vasa deferentia 6–14 µm diameter, posterior vas deferens forms a loop in XI before passing to X; junction to the atrial ampulla median to subapical ( Fig 4A–D ). The ratio of total length of atrium to the body diameter in X is 0.22–0.43 (mean=0.27). Total length of atrium (50 (a)) 63–112 μm, and 32–75 μm wide; atrial ampulla spherical to oval; a short, broadly-conical atrial duct (23–42 µm long) narrows gradually to the male pore, and is about the length of the porophore. In some individuals, the atrial duct forms a short penis by the extrusion of the inner cells (type-2 penis: Rodriguez & Giani 1994) that may protrude through the male pore (to 50 µm) ( Fig. 4D ). Epithelial cells of the ampulla of variable height (4–22 µm); atrial musculature very narrow (c. 2–3 µm). Apical part of the atrial ampulla covered by long prostatic cells (34) 54–104 µm high), usually forming long, tightly packed clusters.

Spermathecae paired in XI and XII, filled with unordered sperm. Spermathecal ampulla usually in the same segment as the corresponding spermathecal pore, although in several individuals some or all spermathecae may penetrate the following segment. The ental region of the ampulla is usually vacuolated and in some cases the sperm is clearly visible inside the vacuoles; it is usually narrower than the ectal section and it may be in the process of degenerating. Spermathecal ducts short (28–72 µm long with a narrow, ental neck (9–12 µm diameter), widening ectally to 21–27 µm ( Fig. 4E ). Spermathecal ampulla irregular, sac-like or tubular, well separated from the duct and variable in size (88–255 µm long, 30–120 µm wide); folded backwards or straight, in a dorsoventral orientation.

Remarks. Among the Trichodrilus species with 2 pairs of spermathecae, T. tenuis Hrabě, 1960 is one of the smallest worms, with a small atrium (usually about one third the body diameter or less), a thin atrial musculature (c. 2 µm) and a cover of high prostatic cells. In the present study, data on the morphology of the Iberian metapopulation are given for the first time, and the general morphological characteristics are compared with those in the type collection of the species (from the NMP) ( Table 3). All characters in the Spanish metapopulation fit the dimensions of the type collection, except for the position of the pharyngeal glands. In the original description of T. tenuis, Hrabě discussed the difference of this species from similar Trichodrilus species ( T. moravicus Hrabě, 1937 and T. allobrogum Claparède, 1862 ). In addition to the small size, Hrabě highlighted that in T. tenuis the pharyngeal glands (“chromophile cells”) are restricted to segments IV– VI, while they extend to VIII in the other two species ( Table 3). In the Spanish collection of T. tenuis , the position of the pharyngeal glands extends to VII or VIII. The examination of Hrabě’s collections of T. tenuis confirms that the position of pharyngeal glands is always in segments IV– VI. Cook (1967: 363) seemed not to be convinced of the taxonomic importance of this character and pointed that this is a variable character in other lumbriculids. In the same way, Rodriguez & Giani (1994) reported some Trichodrilus species showing variability in the extension of the pharyngeal glands, e.g. T. macroporophorus Hrabě, 1954 (back to VII or VIII) or T. pragensis Vejdovský, 1876 (to VIII or IX). It is, however, consistent in the Czech populations of T. tenuis , and may be a useful morphological character in future taxonomic analyses of the species, based on molecular studies.

For the present identification of Trichodrilus tenuis , we emphasize characters of the reproductive system as seen in the type series: an oval, small atrial ampulla ( Fig. 5A–C ) where the vasa deferentia join in a median to subapical position ( Fig. 5B–D ), and a short, conical atrial duct, not petiolated (that is, duct is not well separated by a constriction from the ampulla), which opens in a small, round porophore ( Fig. 5A ). In some individuals a type-2 penis extends through the male pore ( Fig. 5C, E ), perhaps depending on the strength of the fixation, since it was also observed in an unmated individual. Spermathecae have a short duct, bulbous at the ectal end, and ampullae may be straight in the same segment as the pore, or bent and passing to the next segment ( Fig. 5F ).

The spermathecae of T. tenuis were described in detail by Juget & des Chatelliers (2001) from a large collection of several hundred individuals from groundwaters in the region of Lyon, East France. These authors described “ a pseudovestibule with an inner epithelium of conspicuous high cells arranged in a circle around the zone of junction between the duct and the ampulla ”. In our collection, that part of the ampulla close to the duct has a columnar epithelium, and we don’t see anything resembling a vestibule. The more strongly stained (most recent) sperm cells are usually restricted to the ectal section of the spermathecal ampulla, while in French worms the sperm appears distributed along the odd ampulla, which has a thick wall with many constrictions (see Juget & des Chatelliers 2001: fig. 4). In their description, Juget & des Chatelliers also stated that the spermathecal ampullae were always in the same segment as the pore. However, in both Hrabě’s and the Spanish collection, the ampulla may cross to the following segment or not, and the spermathecal ampullae appear either standing up to the dorsal region of the body, as Juget & des Chatelliers (2001) described, or folded on the ventral side toward the posterior part of the segment. All of these differences suggest that the French population of T. tenuis may be a different species.

Habitat and distribution. Groundwaters in the Iberian Peninsula host several populations which have been attributed in present study to the lumbriculid species T. tenuis Hrabě. These sites are up to 700 km distant within Spain and more than 1500 km from the southern and western regions of Germany, where the species was first reported (see Fig. 6 ). The morphospecies T. tenuis is Euro-Mediterranean. In Central Europe, the species distribution covers the High Rhine and Ruhr rivers in Germany ( Juget & Dumnicka 1986). In the Mediterranean region, it has already been reported in groundwaters of eastern France ( Juget & des Chatelliers 2001), Slovenia ( Giani et al 2011), and Spain (wells in Velamazan, Soria: Rodriguez & Giani 1994; Ojo Guareña Cave, Burgos: Rodriguez-Noriega 2013; cave rivers and springs in Bizkaia and Cantabria: Achurra et al. 2015). Unpublished records were also reported from wells in the Rif ( Morocco) ( Juget & des Chatelliers 2001), although these were not included in the guide on the aquatic oligochaetes of Maghreb by Martin & Aït Boughrous (2012). The present study extends the range of distribution of T. tenuis to practically the entire karstic carbonate region in the Iberian Peninsula ( Fig. 6 ), and its habitat covers all types of groundwaters: caves, hyporheos and phreatic waters in wells. However, we are aware that future molecular studies may demonstrate the existence of cryptic speciation in this groundwater species.

Figures

FIGURE 4. Trichodrilus tenuis Hrabě, 1960, from Spanish populations. A–D, atrium. A, Coventosa Cave. B, Ermita San Isidro, Huelva. C, Cullalvera Cave. D, Ojo Guareña Cave. E, Spermatheca from Velamazan Well. A, whole-mounted individual, B–E from dissected worms.All scales represent 50 µm. Some photos are formed by the juxtaposition of two photos, one of them with marked edges, since the atrial duct is usually in another plane than the atrial ampulla.

FIGURE 5. Trichodrilus tenuis Hrabě,1960, syntypes. A, B, D, atrium in Hr 1517-7 (#1). C, atrium and penis Hr1772 (#19). E: Hr 1517-9 (#4) atrium and penis. F, spermatheca Hr 1517-7 (#1). All sagittal histological sections, except for E, from a wholemount. All scales 50 µm.

FIGURE 6. Distribution map of Trichodrilus tenuis (in red) and T. moravicus (in green) (from Timm & Abarenkov), and Iberian T. tenuis from the present study (in black). Karst areas of Spain (Oliveira et al. 2009) in the upper left square, marked in black.