Species only known, until now, from Gotland, Sweden (Larsson et al. 2008; Larsson and Willems 2010).

Two specimens studied alive and stored in absolute ethanol for molecular analyses, one of them sequenced; collected in Kirchwerder-Fünfhausen, submerged vegetation and litter in an irrigation channel, 0.1–0.2 m deep.

Specimens measuring 768–957 µm long (x̄ = 863 µm; n = 2) and 90–110 µm at widest point (x̄ = 100 µm; n = 2), with two zooids, slender, tapering to both rounded extremes (Fig. 1A). The ciliated pits (Fig. 1B: cp) are relative short and open close to the most anterior part of the body. The epidermis is fully ciliated. Larger cilia are distributed along the body, particularly in the anterior and posterior ends. The brain (Fig. 1B: br) consists of two pairs of lobes, the anterior brain (Fig. 1C: ab) and the posterior brain (Fig. 1C: pb). We were not able to determine the exact number of compartments of the anterior brain, but in one specimen there appear to exist seven. Refractile bodies not present. Proximal rim of the pharynx (Fig. 1A, B: ph) with a number of folds and surrounds the large mouth opening (Fig. 1A, B: m). The protonephridium (Fig. 1D: pn) ends in a nephridiopore at the posterior end of the body.

Figure 1.

Stenostomum gotlandense. A. Habitus of a swimming specimen; B. Anterior part of the body; C. Anterior part of the body showing the brain; D. Posterior part of the body. Abbreviations: ab anterior brain; br brain; cp ciliated pits; ep excretophore; i intestine; m mouth; pb posterior brain; ph pharynx; pn protonephridia. Scale bars: 50 μm (A, C); 100 μm (B, D).

As noted by Larsson and Willems (2010), the folded rim of the pharynx, the large mouth opening, and the small ciliated pits represent a unique combination of morphological traits within Stenostomum. Our morphological identification is further corroborated by the phylogenetic analysis, leading us to confidently report this species for the first time in Germany, specifically in Hamburg. The German specimens are similar in size, considering the length of the first zooid (495–588 µm), to those from Sweden (500 µm). However, Larsson and Willems (2010) observed that their specimens exhibited approximately 10 compartments in the anterior brain, whereas the specimens from Hamburg present about seven. Nonetheless, one specimen illustrated by Larsson and Willems (2010: fig. 3B) shows only six segments in the anterior brain.

Species with a broad distribution through North America (United States and Mexico) (Higley 1918; Nuttycombe and Waters 1938; Kolasa et al. 1987; Núñez-Ortiz et al. 2016; Glasgow 2021), South America (Argentina, Brazil, Peru, and Suriname) (Marcus 1945; van der Land 1970; Noreña-Janssen 1995; Gamo and Leal-Zanchet 2004; Noreña et al. 2005; Damborenea et al. 2011; Reyes et al. 2021), Iceland and Faroe Islands (Steinböck 1948), West Europe (United Kingdom, Ireland, The Netherlands, Germany, Switzerland, and Spain) (Graff 1882; Hofsten 1911; Gieysztor 1931; Young 1970, 1972, 1973; Noreña et al. 2007), East Europe (Poland, Romania, Serbia, and Croatia) (Graff 1882; Steinböck 1933; Kolasa 1971; Mack-Fira 1974), Russia (Nasonov 1926; Steinböck 1932; Timoshkin et al. 2010), Asia (Thailand and Japan) (Yamazaki et al. 2012; Ngamniyom and Panyarachun 2016), and Africa (Tanzania and Kenya) (Young and Kolasa 1974; Young 1976).

Three specimens studied alive and stored in absolute ethanol; collected in Kirchwerder-Fünfhausen, submerged vegetation and litter in an irrigation channel, 0.1–0.2 m deep. Three specimens studied alive and stored in absolute ethanol; collected in Groß Glienicker lake, littoral, floating vegetation. Three specimens studied alive and stored in absolute ethanol; collected in Sylt, floating vegetation in a small pond. Two specimens from Kirchwerder-Fünfhausen and one from Sylt were sequenced for the molecular analyses.

Specimens measuring 1120–1490 µm long (x̄ = 1305 µm; n = 2) and 175–220 µm at widest point (x̄ = 198 µm; n = 2), with two zooids, anterior end rounded and posterior tapering (Fig. 2A–C). The ciliated pits (Fig. 2D: cp) are relative short and open close to the most anterior part of the body. The epidermis is fully ciliated. Larger cilia are distributed along the body, particularly in the anterior and posterior ends. The brain (Fig. 2A, B, D: br) consists of two pairs of lobes, the anterior brain (Fig. 2F, G: ab) and the posterior brain (Fig. 2F, G: pb). The anterior brain is more distinct but does not show a clearly compartments. A pair of refractile bodies (Fig. 2F, G: rb) are connected to the posterior brain via stalked structures. The refractile bodies are 8–9 µm in diameter (n = 3). The number of spherules contained in the refractile bodies is difficult to observe but they are more than 20 in all studied specimens. The refractile bodies can appear either rounded or crescent-shaped, depending on the orientation of these structures.

Figure 2.

Stenostomum leucops. A–C Habitus of swimming specimens; D. Anterior part of the body; E. Posterior part of the body F, G. Anterior part of the body. Abbreviations: ab anterior brain; br brain; cp ciliated pits; i intestine; m mouth; np nephridiopore; pb posterior brain; ph pharynx; phg pharyngeal glands. Scale bars: 200 μm (A–C); 100 μm (D, E); 50 μm (F, G).

Our identification of specimens was primarily based on Luther’s (1960) description. However, recent studies have suggested that S. leucops may actually represent a complex of cryptic species, possibly related to S. grande Child, 1902 (see Yamazaki et al. 2012; Rosa et al. 2015). Molecular evidence supports this notion and echoes earlier discussions by Nuttycombe and Waters (1938) and Marcus (1945), who both deemed the available description insufficient for clear recognition of the species. Rosa et al. (2015) identified three distinct clades within S. leucops, each distributed in Brazil, the United Kingdom, and Sweden, respectively, but were unable to find morphological diagnostic traits to differentiate them. The phylogenetic analysis here developed (see section Molecular phylogenetic analyses) shows a close relationship between S. leucops from Germany and Sweden.

Given that the type locality of S. leucops is in the vicinity of New York, United States, a comprehensive morphological and molecular phylogenetic analysis of specimens from that area is essential to stabilize the classification of S. leucops (see Discussion).

Species with a broad known distribution including Europe (United Kingdom, The Netherlands, Germany, Finland, Spain, and Italy) (Papi 1951; Luther 1960; Rixen 1961; Young 1970, 1973; Tulp 1974; Farias et al. 1996; Noreña et al. 2007), Russia (Luther 1960), and Kenya (Young 1976).

Two specimens studied alive, one preserved in ethanol for future molecular analyses, the second cut in two pieces, the posterior part containing the stylet for whole mounting and the anterior part preserved for molecular analyses. Collected in Wandse river, submerged vegetation with organic matter, 0.1 m deep.

Description. Animals 0.8–0.9 mm long (n = 2), unpigmented and with a pair of eyes (Fig. 3A, B: e). General morphology corresponds to that in previously described specimens (see Papi 1951). Epidermis fully ciliated, with some larger cilia distributed though the body. False seminal vesicle absent and true seminal vesicle (Fig. 3C–E: sv) opening into the prostate vesicle (vesicula granulorum). The prostate vesicle (Fig. 3C–F: pv) opens proximally into the stylet (Fig. 1A, C–F: st). One specimen had a fully developed stylet, measuring 60 µm in length; it is hook shaped and with the distal part twisted; aperture subdistal and dorsal. Well-developed eggs observed (Fig. 3A, C: eg).

Figure 3.

Macrostomum rostratum. A. Habitus of a swimming specimen; B. Anterior part of the body; C–F. Posterior part of the body. Abbreviations: e eye; eg egg; ph pharynx; pv prostate vesicle; st stylet; sv seminal vesicle. Scale bars: 200 μm (A); 50 μm (B–F).

According to the drawings of Papi (1951: figs 32–33) the stylet of this species measures 42–65 µm, which is in the range of our collected specimen. In other populations recorded from Germany, the stylet is 65–77 µm long (Rixen 1961). The most characteristic traits for this species are the distal turn and the dorsal aperture of the stylet; therefore, we identify our specimen as M. rostratum.

Species with a worldwide distribution, recorded from South America (Brazil) (Marcus 1944; Reyes et al. 2021), North America (Unite States) (Higley 1918; Kenk 1949; Kolasa et al. 1987; Smith 1991; Glasgow 2021), Faroes Islands (Steinböck 1931), West Europe (United Kingdom, Ireland, Sweden, Belgium, Netherlands; Germany, Switzerland, Austria, Poland, Czech Republic, France, Spain) (Schultze 1851; Graff 1882, 1913; Fuhrmann 1894, 1900; Steinböck 1923; Southern 1936; Rixen 1961; Young 1970, 1972, 1973; Kolasa 1971; Farias et al. 1996; Noreña et al. 2007), East Europe (Bulgaria, Hungary, Hungary, North Macedonia-Alvania, Russia) (Graff 1913; Steinböck 1932; An der Lan 1939), Asia (China and Japan) (Okugawa 1953; Peng et al. 2007), and Africa (Kenya) (Young and Young 1976).

Six specimens studied alive, two preserved in ethanol for future molecular analyses, four cut in two pieces, the anterior part containing the stylet for whole mounting (ZMH V13839–13842) and the posterior part preserved for molecular analyses; collected in Kirchwerder-Fünfhausen, submerged vegetation and litter in an irrigation channel, 0.1–0.2 m deep.

Animals 2.5–3.5 mm long depending on the contraction stage, opaque, without eyes (Fig. 4A). The oral pore opens at the anterior tip; the pharynx (Fig. 4B, C: ph) is located behind the brain (Fig. 4B: br) and beside the prostate vesicle (Fig. 4B, C: pv), and proximally opens into the intestine (Fig. 4A: i). The intestine of some specimens contained numerous chaetae of oligochaetes; a feeding behaviour previously recorded to the species (Tylet et al. 2018).

Figure 4.

Prorhynchus stagnalis. A. Habitus of a swimming specimen; B–D. Anterior part of the body; E, F. Sclerotised structures. Abbreviations: br brain; eb external bars; i intestine; ib internal bars; ov ovary; ph pharynx; phg pharyngeal glands; pv prostate vesicle; st stylet. Scale bars: 200 μm (A); 100 μm (B–D); 50 μm (E, F).

The ovary (Fig. 4A: ov) is very long and runs medially, extending over the posterior two thirds of the body; eggs were observed. The male reproductive system includes the large unpired testis, located at mid body; the seminal vesicle, located posterior to the pharynx; the prostate vesicle running beside the pharynx; and the armed copulatory organ. The prostate vesicle shows thick muscle walls, perforated by the necks of the extracapsular prostatic glands, containing a coarse-granular secretion. Proximally, the prostate vesicle looks empty, maybe because it contains a very fine secretion. A very large duct connects the prostate vesicle with the armed bulb.

The armed male copulatory organ (Fig. 4D–F) is located in the anterior part of the body, anterior to the brain. It consists of a central stylet (Fig. 4B, D–F: st) surrounded by two concentrical rings of spines. The stylet is 64–68 µm long (x̄ = 66 µm; n = 4), proximally funnel shaped and posteriorly tapering to the distal sharp tip. As described by Tyler et al. (2018), the stylet consist of two pieces, clearly distinguishable in the posterior third of the stylet. In the distal area, only the sharp tip of the stylet (part of the inner tube) could be observed. The number of bars on each ring surrounding the stylet is difficult to observe; however, in two specimens it seems that there are 10 bars in the external and six in the internal ring. This pattern has also been identified in populations from the Unite States and Brazil (Tyler et al. 2018; Reyes et al. 2021). The external bars (Fig. 4D–F: eb) are arched, 53–57 µm long (x̄ = 55 µm; n = 4), proximally broad and posteriorly taper to rounded tips. The internal bars (Fig. 4D, F: ib) appear straight to slightly arched but the rest of their morphology is hardly distinguishable; they are 47–53 µm long (x̄ = 50 µm; n = 4).

It has been proposed that P. stagnalis comprises a complex of species primarily distinguished by variations in the morphology of male sclerotised structures. However, comprehensive morphological and molecular investigations are imperative to elucidate the taxonomy of this species group fully. Furthermore, extensive sampling is required, including the type locality of the species (Greifswald, Germany). The specimens we have examined exhibit the smallest stylet (~66 µm) compared to those documented in the literature from The Urals (100–124 µm; Rogozin 2015), the United States (~90 µm; Tyler et al. 2018), and Brazil (~123 µm; Reyes et al. 2021).

This is the microturbellarian species with the widest distribution worldwide (see Artois and Tessens 2008; Diez et al. 2018). However, this distribution corresponds to a puzzle of dozens of cryptic species (Tessens et al. 2021).

Specimens were found in two locations. One specimen was studied alive and preserved in ethanol for future molecular analyses. It was collected in Wandse river, submerged vegetation with organic matter, 0.1 m deep. Fifteen specimens were studied alive, eleven of them whole mounted and four preserved for future molecular analyses collected in Kirchwerder-Fünfhausen, among submerged vegetation and litter in an irrigation channel, 0.1–0.2 m deep.

The specimen from Wandse river is 1354 µm long and those from Kirchwerder-Fünfhausen are 870–926 µm long (x̄ = 904 µm; n = 6). They are unpigmented, with a pair of eyes (Fig. 5A: e) posterior to the proboscis (Fig. 5A: pr).

Figure 5.

Gyratrix hermaphroditus. A, B. Habitus of a swimming specimen; C–F. Posterior part with sclerotised structures. Abbreviations: b bursa; e eye; ov ovary; ph pharynx; pr proboscis; ps3, ps4 prostate stylet type 3 and 4, respectively; pv prostate vesicle; t testi. Scale bars: 100 μm (A); 50 μm (B–F).

The testis (Fig. 5A, B: t) is located at the left body side, extending from the region of the eyes to about three quarters of body length. Ovary and atrial organs located posteriorly in the body. The prostate stylet type II (Fig. 5B–F: ps2) is 189 µm long in the specimen from Wandse river and 155–169 µm long (x̄ = 164 µm; n = 9) in those from Kirchwerder-Fünfhausen. The prostate stylet type III (Fig. 5B–F: ps3) is 147 µm long in the specimen from Wandse river and 150 µm long (x̄ = 131–167 µm; n = 9) in those from Kirchwerder-Fünfhausen. In the specimens from both localities, the stylet type III distally bifurcates and therefore ends in two opposed tips.

Vitellarium not observed. The ovary (Fig. 5B, C: ov) is kidney shaped in the specimen from Wandse river, with the oocytes organised in a row, increasing in diameter from proximal to distal. In contrary, in the specimens from Kirchwerder-Fünfhausen, the ovary is globular, with oocytes of dissimilar size. The bursa (Fig. 5B–D: b) is located completely posterior and overlaps with the stylets; several masses of sperm in digestion were observed.

Based on our findings, it appears that the specimens collected from the two sampled localities correspond to two distinct species within the G. hermaphroditus complex. These species are distinguishable by differences in the size of the sclerotised male structures and the morphology of the ovary. However, differentiation within this extensive species complex poses significant challenges and warrants molecular investigations. Phylogenetic analyses conducted by Tessens et al. (2021) revealed a remarkable diversity of 62–78 species concealed under the name G. hermaphroditus, with many of them confined to single localities while others are more widely distributed.

The morphology of the hard structures in our specimens appears to align with group H as defined by Tessens et al. (2021), characterised by the bifurcate ending of prostate stylet type III. Notably, this group comprises exclusively freshwater species from Australia. However, molecular studies are essential to validate this morphological resemblance. Specimens from Germany included in the aforementioned phylogenetic study fall into groups A and B (comprising freshwater and brackish species) and group L (marine species). Nonetheless, representatives from the latter three groups exhibit a prostate stylet type III ending in a simple, hook-shaped tip. Future integrative studies are imperative to unravel the intricate taxonomy of the G. hermaphroditus species complex, which should encompass the analysis of specimens from its type locality in Berlin, Germany.

Species recorded from Finland and Sweden (Luther 1955).

Seven specimens studied alive, two of which were whole mounted afterwards (ZMH V13831–13832); collected in Kirchwerder-Fünfhausen, submerged vegeta­tion and litter in an irrigation channel, 0.1–0.2 m deep. Four specimens studied alive and preserved in absolute ethanol, collected in Groß Glienicker lake; littoral, floating vegetation. Two specimens from Kirchwerder-Fünfhausen and one from Groß Glienicker lake used for molecular phylogenetic analyses.

Live specimens about 1 mm long, anterior margin rounded and posterior pointing, translucent, pinkish-brownish colouration due to parenchymal glands (Figs 6–7). A pair of eyes (Figs 6A–C, 7A: e) anterior to the pharynx; the eyes are formed by two pigmented spots linked by a bridge, and sometimes look like two independent structures. Barrel-shaped pharynx (Figs 6A–C, 7A: ph) 122–183 µm long (x̄ = 147 µm; n = 5). Testes located postero-lateral to the pharynx. The male copulatory organ includes a seminal vesicle (Fig. 7E, F: sv), a prostate vesicle (Fig. 7E, F: sv), and the stylet. The stylet (Figs 6D–F, 7C–F: st) is 43–74 µm long (x̄ = 60 µm; n = 3) and displays a proximal handle and a distal spiny part; a hook-shaped, well-differentiated, largest spine is 14–30 µm long (x̄ = 22 µm; n = 3). The vitellaria (Fig. 6B: vi) run beside the pharynx until the posterior third of the body, fusing before opening into the oviduct. The oval eggs are 64–120 µm long (n = 2) and present a 95–168 µm long stalk (n = 2) (Fig. 7A–C: eg).

Figure 6.

Castrella alba Luther, 1955 collected in Hamburg. A, B. Habitus of a swimming specimen; C. Details of the anterior part of the body; D–F. Posterior part of the body with atrial structures. e eye; ov ovary; ph pharynx; st stylet; vi vitellaria. Scale bars: 50 μm.

Figure 7.

Castrella alba Luther, 1955 collected in Brandenburg. A. Habitus of a swimming specimen; B. Details of an egg and its stalk; C. Details of an egg and the stylet; D–F. Posterior part of the body with atrial structures. e eye; eg egg; egs egg stalk; ph pharynx; st stylet; vi vitellaria. Scale bars: 50 μm.

As noted by Luther (1955), identifying the male sclerotised apparatus in C. alba is particularly challenging. In our study, we were able to distinguish a fully developed stylet in only three out of 11 specimens, while this structure was indistinguishable in the whole mounted material. The stylet in this species is generally delicate and, at first glance, appears underdeveloped. Consequently, Luther (1955) speculated that this species might be synonymous with C. vernalis Beklemischev, 1921. However, the newly studied material, which includes fully developed specimens, suggests that C. alba bears a closer resemblance to C. truncata (Abildgaard, 1789) Sekera, 1906, as indicated by the stylet, which has a proximal handle and a distal spiny section with one spine more developed than the others. Nevertheless, our phylogenetic analysis confirms that C. alba represents a distinct lineage from C. truncata (see section Molecular Phylogenetic Analyses; Fig. 16).

This species has been broadly recorded in the United States, Iceland, Faroe Islands, overall Europe (United Kingdom, Ireland, Finland, Belgium, Germany, Austria, Switzerland, Czech Republic, Poland, Romania, Bulgaria, Latvia, Italy, France, and Spain), Russia, and Japan. See a summary of this distribution and main references in Van Steenkiste et al. (2011).

One specimen studied alive and whole mounted (ZMH 13833), collected in Wandse river, submerged vegetation with organic matter, 0.1 m deep.

Live animal about 1 mm long, anterior margin rounded and posterior pointing, translucent, orange colouration due to parenchymal glands (Fig. 8A). A pair (Fig. 8A: e) of eyes anterior to the pharynx. Barrel-shaped pharynx (Fig. 8A: ph) 250 µm long. Testes located antero-lateral to the male copulatory bulb. This bulb encompasses a seminal vesicle (Fig. 8B: sv), a prostate vesicle (Fig. 8B: sv), and the stylet. The stylet (Fig. 8C–E: st) is 123 µm long, H-shaped as typical for species of Microdalyellia. One arm carries five 17-µm-long spines, and the other a single 58-µm-long spine. The vitellaria (Fig. 8A: vi) run beside the pharynx until the posterior third of the body, fusing before opening into the oviduct. The oval egg is 140 µm long (Fig. 8A, C–F: eg).

Figure 8.

Microdalyellia armigera. A. Habitus of a swimming specimen; B. Seminal and prostate vesicles; C–F. Male stylet. e eye; eg egg; i intestine lumen; ph pharynx; pv prostate vesicle; st stylet; sv seminal vesicle; vi vitellaria. Scale bars: 100 μm (A); 50 μm (B–F).

Microdalyellia armigera shows a high morphological variability of the male sclerotised stylet, the main character to differentiate species in most microturbellarians. However, it would not be surprising if this variability represents cryptic speciation, as suggested by our phylogenetic analysis (see section Molecular Phylogenetic Analyses; Fig. 16). For example, the specimens of M. armigera collected by Müller and Faubel (1993) in the Elbe river present a stylet much larger than in our specimen (220 µm vs 123 µm respectively) and have 3–9 spines in one arm. The size of this structure in other North-European populations varies between 82 μm and 130 μm (Luther 1955; Rixen 1961) and in specimens from Spain is 165 μm long (Van Steenkiste et al. 2011). In general the number of spines in both arms can be variable (see Luther 1955; Bauchhenss 1971) but commonly there is one arm with one spine and the other with 4–5 (Müller and Faubel 1993).

Species with a known distribution in West Europe (United Kingdom, Finland, Germany, and Switzerland) (Graff 1882, 1913; Fuhrmann 1894; Luther 1955; Rixen 1961; Young 1970), and Russia (Nasonov 1919, 1926).

Observations on eight live animals, five whole mounted afterwards (ZMH 13834–13838) and the other three preserved in absolute ethanol (two already sequenced for molecular analyses); collected in Wittenberg, Rissen, on tree holes filled with water and litter, 20–50 cm over the ground level.

Animals 704–1010 µm long (x̄ = 840 µm; n = 3), with a pair of eyes (Fig. 9A: e) anterior to the pharynx. The yellowish to orange colouration is due to parenchymal glands. The barrel-shaped pharynx (Fig. 9A: ph) is 231–289 µm long (x̄ = 262 µm; n = 3), when the animal is not contracted.

Figure 9.

Microdalyellia schmidtii. A. Habitus of a swimming specimen; B, C. Male atrial organs; D. Ovary; E, F. Male stylet. Scale bars: 100 μm (A); 50 μm (B–F).

The atrial organs are located in the posterior body third. The pair of testes (Fig. 9B, C: t) is located beside the male copulatory organ. The copulatory organ encompasses a seminal vesicle (Fig. 9B, C: sv), a prostatic vesicle (Fig. 9B, C: pv), and the stylet. The stylet (Figs 9B, C: st, E, F, 10) is 147–151 µm long (x̄ = 149 µm; n = 4) and each arm is armed with a conical spine. The larger spine is 64–75 µm long (x̄ = 69 µm; n = 4) and the smaller is 48–52 µm long (x̄ = 50 µm; n = 4), both with the distal tip bent outside and hook shaped. Each vitellarium (Fig. 9A: vi) runs beside the pharynx until the posterior third of the body, and fuses before entering the oviduct. The ovary (Fig. 9D: ov) is 157–212 µm long (x̄ = 185 µm; n = 2). The distal most three oocytes are of similar size and organised in a row, the rest of the ovary is full of smaller oocytes with different degrees of development. The eggs (Fig. 9A: ov) are oval, 148–206 µm long (x̄ = 177 µm; n = 4).

Around 44 species of Microdalyellia have been recorded globally (Tyler et al. 2006–2024). Among them, M. schmidtii stands out as the only one possessing a single, funnel-shaped spine on each arm of the stylet, leading to its stylet being described as ‘plow-shaped’ (Graff 1882; Luther 1955). However, doubts have been raised regarding the validity of this species due to its similarity to M. armigera and M. kupelwieseri (Meixner 1915; Ruebush and Hayes 1939). It has been suggested that M. schmidtii and M. kupelwieseri might be forms of M. armigera with a greatly reduced number of spines (Luther 1955; Young 1970).

Nasonov (1919, 1926) provides insight into the close relationship among the previously mentioned species, particularly in Russian populations. Some specimens of M. schmidtii from Russia (Nasonov 1919: fig. 8) resemble M. kupelwieseri due to the presence of a few smaller spines alongside the main spine of one arm (see Meixner 1915; Luther 1955; Young 1970). However, Nasonov (1926) also recorded specimens of M. schmidtii with a single spine per arm, some displaying transitional forms between extreme morphologies of M. schmidtii and M. armigera. Nevertheless, populations of M. kupelwieseri from Germany (Rixen 1961), the United Kingdom (Young 1970), and Spain (Farías et al. 1995) show morphological convergence, with stylets measuring 100–110 µm and carrying one spine in one arm and three in the other.

The Hamburg population of M. schmidtii is notable for its significantly larger stylet (140 µm) compared to other recorded populations: 92 µm in the United Kingdom (Young 1970), 66 µm in Finland (Luther 1955), and 100 µm in Germany (Rixen 1961). Stylet sizes in M. kupelwieseri populations are also smaller (100–110 µm). The stylet in the new studied material l (ZMH V13848– V13856) of M. kupelwieseri populations displays a triangular spine in the bridge linking the two branches of the stylet (Fig. 11B, D: sp), a structure lacking in both M. schmidtii and M. armigera. In addition, one of the spiny arms of M. kupelwieseri carries three spines, whereas the number is always one in M. schmidtii from Hamburg. We suspect, therefore, that the high similarity between the two species and the sympatric distribution of both in some localities, contributed with their misidentification. Indeed, the re-examination of two specimens identified as M. kupelwieseri collected in Belgium and stored in the reference collection of Hasselt University (VII.4.20 and VII.4.24) allowed their reclassification as M. schmidtii. This action is made considering that their stylets lack a triangular spine in the bridge. A third specimen collected in Belgium, together the two previously mentioned, and sharing the same morphology, was included in the phylogenetic analysis by Van Steenkiste et al. (2013) and it clustered in our phylogeny with specimens of M. schmidtii collected in Hamburg (see section Molecular Phylogenetic Analyses; Fig. 16). These findings support that the presence or not, of a triangular spine in the stylet’s bridge is a diagnostic character for M. kupelwieseri and M. schmidtii, respectively.

Figure 10.

Microdalyellia schmidtii. A–F. Male stylet. Scale bars: 50 μm (A–F).

Figure 11.

Microdalyellia kupelwieseri. A–F. Male stylet; D. Detail of the bridge’s spine; E–F. Detail of the spiny branches. Scale bars: 50 μm (A–F).

Until now, the relationships among M. armigera, M. kupelwieseri, and M. schmidtii, and the validity of the latter two species, remained unclear. However, it is suggested that the group M. kupelwieseri – schmidtii can be distinguished from M. armigera by the reduced spine number in one arm, as well as the larger size of the single spines in M. schmidtii and the main spines of M. kupelwieseri compared to those in M. armigera. In this sense, our phylogenetic analysis contributed to clarify that the three species represent distinct lineages and support their validity. However, M. armigera could represent a complex of cryptic species (see section Molecular Phylogenetic Analyses; Fig. 16).

Until now, this species was only known from its type locality in Schlitz, Hessen, Germany (Schwank 1979).

Sixteen specimens studied alive, five of them whole mounted (ZMH V13843–13847), nine preserved for future histological, and two used for molecular studies. Animals collected in Wittenberg, Rissen, on tree holes filled with water and litter, 20–50 cm over the ground level.

Live animals 1.5–2 mm long, unpigmented and without eyes (Fig. 12A). Strong adhenal rhabdite tracts (Fig. 12B: ar) open in the anterior body end. Body oval-elongated, with the anterior margin broader than the posterior. The pharynx (Fig. 12A, C: ph) is located about midbody, 285 µm in diameter (all provided measures of this species are based on a single living specimen).

Figure 12.

Krumbachia hiemalis. A. Habitus of live specimens; B. Anterior region; C. Posterior region; D, E. Atrial organs. Scale bars: 50 μm (B–E).

The testes (Fig. 12A: t) are located anterior and the ovary and atrial organs posterior to the pharynx. The male copulatory organ (Fig. 12A, C: mco) is 156 µm long and encompasses a seminal vesicle, a prostate vesicle, the sclerotised ejaculatory duct, and a bundle of accessory glands. The seminal vesicle (Fig. 12D, E: sv) receives independently both spermatic ducts and, then, opens into the prostate vesicle. The prostate vesicle (Fig. 12D, E: pv) contains a granular secretion, also observable throughout the ejaculatory duct. The ejaculatory duct (Fig. 12D, E: ed) is 102 µm long, with slightly sclerotised walls (not observable on the whole mounts). The distal half of the male copualatory bulb is occupied by a mass of accessory glands (Fig. 12D, E: gl), surrounding the ejaculatory duct.

The vitellaria (Fig. 12A: vi) extend at the body sides, between the brain and the posterior part of the body. The ovary (Fig. 12A, C: ov) is 152 µm long, with the oocytes increasing in diameter from proximal to distal. The female duct connects the ovary to the female atrium. A seminal reservoir (Fig. 12A: sr) opens into the female duct (no sperm observed in this structure). The bursa (Fig. 12A, C–E: b) opens into the common general atrium in between the apertures of the female and male atria. Several vesicles were observed within the bursa but no sperm.

The morphology of the specimens collected in Hamburg closely aligns with the description outlined by Schwank (1979). For specimens from the type locality, the copulatory bulb length falls within the range of 120–180 µm. Additionally, Schwank (1979) noted the proximal oblique opening of the sclerotized duct in this species, a characteristic clearly observable in our recently collected specimens. This species stands out from all others within the genus Krumbachia due to the distinctive combination of a two-layered cuticularised ejaculatory duct, notable for its high mobility, and a bursa characterised by a remarkably thin wall.

Species broadly distributed in West Europe (United Kingdom, Ireland, Belgium, Finland, Sweden, Denmark, Germany, Greece, France, and Italy) (Arndt 1926; Luther 1961; Ronneberger 1975; Ball and Reynoldson 1981; Müller and Faubel 1993; Martin and Brunke 2012), East Europe (Estonia, Latvia, Littauen, Ukraine, Poland, and Czech Republic) (Luther 1961; Pinchuk 1979), and Russia (Grimm 1877; Luther 1961).

Six specimens studied alive and preserved in absolute ethanol for future molecular analyses; one collected in Wandse river, submerged vegetation with organic matter, 0.1 m deep; one in Planten un Blomen park, submerged litter, 0.3 m deep; and four in Kirchwerder-Fünfhausen, submerged vegetation and litter in an irrigation channel, 0.1–0.2 m deep.

Live adult specimens measuring 0.5–1.5 cm, dark pigmented, with a pair of anterior eyes (Fig. 13A–C: e). Squeezed specimens show the pharynx and atrial organs. The pharynx (Fig. 13C, D: ph) is located in the second body half and the mouth opens anterior to the male copulatory organ (Fig. 13A, C, D: mco). The seminal ducts form false seminal vesicles (Fig. 13A, C, D: fsv) beside the anterior part of the pharynx. The male copulatory organ receives medially, independently, both seminal ducts, which evacuate the sperm in a single proximal seminal vesicle. Distally, the bulb forms a muscular penial papilla. One adenodactyl (Fig. 13C, D: ad) opens into the common atrium, at the right side of the male bulb; it is oriented backwards. The adenodactyl is distally bent and the central lumen makes it look hollow. The single observed structure of the female system was the bursa (Fig. 13D: b), located to the right side of the male organ.

Figure 13.

Planaria torva. A. Habitus of swimming specimen; B–D. Squeezed specimen.

Planaria torva is a species widely distributed throughout West Europe, and it is frequently mentioned in taxonomic literature on triclads in the region. However, accurate identification of this species can be challenging without a detailed examination of internal morphology. The taxonomic history of P. torva has been contentious, with several studies mistakenly associating it with species of Dugesia (Ball et al. 1969). In light of these issues, we based the identification of our specimens on descriptions provided by Luther (1961) and Ball et al. (1969). The presence of an adenodactyl serves as a key distinguishing feature between our studied specimens of P. torva and species of Dugesia (Luther 1961). Furthermore, the overall structure of the atrial organs in our specimens, particularly the male bulb and the adenodactyl, unequivocally supports their classification within P. torva. For a more comprehensive comparison of this species with related congeners, refer to Ball et al. (1969).

Species recorded from The Netherlands (Young 1972), Finland, United Kingdom and Ireland (Luther 1961), Gernany (Schwank 1981; Martin and Brunke 2012), Macedonia (Kenk 1978), Romania (Felicia 2018), and Russia (Luther 1961).

Six specimens studied alive, preserved in absolute ethanol for future molecular analyses; collected in Kirchwerder-Fünfhausen, submerged vegetation and litter in an irrigation channel, 0.1–0.2 m deep.

Mature specimens measuring 0.5–1.2 mm, dark coloured (Fig. 14A, B). Marginal eyes (Fig. 14C, D: e) distributed over the anterior third of the body. The pharynx (Fig. 14B, C) is located over the midbody and the mouth (Fig. 14C, E: m) opens anterior to the male copulatory organ. The male copulatory organ (Fig. 14B, E: mco) is 940–1100 µm long (n = 1; varying according to the relaxing stage) and 620 µm at widest. The male organ is spiny over its distal 300–480 µm, and forms a penial papilla (Fig. 14E: pp). The spines (Fig. 14F) are 17–18 µm long (n = 10). Two adenodactyls (Fig. 14E: ad) are located posterior to the male bulb and open into the common atrium, oriented forward, and exhibiting a glandular lumen.

Figure 14.

Polycelis tenuis. A. Habitus of swimming adult specimen; B. Squeezed adult specimen; C, D. Squeezed juvenile; E. Atrial organs; F. Spines of the penial papilla. Scale bars: 200 μm (D); 600 μm (E); 100 μm (F).

Three species of Polycelis have been documented in Germany, and they are widespread across Europe: P. felina, P. nigra, and P. tenuis (Volk 1903; Ronneberger 1975; Schwank 1981; Müller and Faubel 1993). Among these, only P. nigra has been reported in Hamburg (Volk 1903). Species within the genus Polycelis are primarily distinguished by the structure of the male bulb and adenodactyls. Polycelis tenuis shares with P. felina the presence of two adenodactyls, structures absent in P. nigra. However, P. felina is easily identifiable by the presence of two tentacles in its anterior body region. The penial papilla of P. tenuis is armed with spines along its distal half, whereas P. nigra exhibits two to three spine rows distally, and P. felina lacks any spines in this region (Hansen-Melander et al. 1954; Luther 1961; Harrath et al. 2012). Volk (1903) did not provide detailed morphological information about the specimens of P. nigra recorded in Hamburg. Given the necessity of studying the morphology of atrial organs for accurate identification of these triclads, this record requires confirmation.