Dugesia benazzii Lepori, 1951

Dols-Serrate, Daniel, Stocchino, Giacinta Angela, Sluys, Ronald & Riutort, Marta, 2024, Fantastic beasts and how to delimit them: an integrative approach using multispecies coalescent methods reveals two new, endemic Dugesia species (Platyhelminthes: Tricladida) from Corsica and Sardinia, Zoological Journal of the Linnean Society 201 (3), pp. 1-29 : 12-17

Dugesia benazzii Lepori, 1951

Features of Dugesia benazzii s.s.: Finger-shaped penis papilla; central course of the ejaculatory duct, opening terminally at the tip of the penis papilla; conical diaphragm; symmetrical openings of the oviducts into the bursal canal; common genital atrium; penial fold devoid of a lumen, localized on the less side to the penis papilla.

Material examined: Individuals from populations 2, 6, 9, 10, 11, 12, 13, 14, and 16 ( Fig. 1 ) were analysed molecularly; GenBank accession numbers are detailed in the Appendix Table A1 . Animals examined histologically were: CGAS Pla 25. 1, Rio su Rizzolu , locality 13, Coghinas River basin (40°43ʹ55ʹʹN, 9°08ʹ36ʹʹE), Sardinia, 28 April 1993, coll. M. Pala and G.A. Stocchino, sagiưal sections on nine slides ; CGAS Pla 25. 2, ibid., sagiưal sections on 12 slides ; CGAS Pla 25. 3, ibid., transverse sections on 15 slides ; CGAS Pla 25. 4, ibid., transverse sections on seven slides ; CGAS Pla 25. 5, ibid., horizontal sections on six slides ; CGAS Pla 25. 6, ibid., sagiưal sections on 20 slides ; RMNH.VER.21055.1, ibid., sagiưal sections on 16 slides ; RMNH.VER.21055.2, ibid., transverse sections on 16 slides. Karyological information was extracted from Vacca et al. (1993). A karyogram of the Su Rizzolu population is here reported for the first time ( Fig. 6A ) .

Diagnosis: Molecularly, Dugesia benazzii comprises individuals that are identified as a single evolutionary unit together with individuals from populations 2, 6, 9, 10, 11, 12, 13, 14, and 16, when using the coalescence-based method BPP with the loci and seưings detailed in the present study’s Materials and methods section or equivalent molecular delimitation analyses. Morphologically, D. benazzii is characterized by the presence of the following features: finger-shaped penis papilla; central course of the ejaculatory duct, opening terminally at the tip of the penis papilla; conical diaphragm positioned at the base of the penis papilla; asymmetrical course of the vasa deferentia into the penis bulb, with the right sperm duct running dorso-laterally to the less one, which runs ventro-laterally; asymmetrical openings of the vasa deferentia into the posterior section of the seminal vesicle, close to the diaphragm; symmetrical openings of the oviducts into the bursal canal; ectal reinforcement confined to the posterior tract of the vaginal area of the bursal canal; angled bursal canal; penial fold of the parenchymatic-muscular type. Karyology: 2n = 16; chromosome pairs all metacentric, except chromosome 7, which is submetacentric.

Morphological redescription: Inner and outer pharyngeal musculature bilayered, i.e. without an extra, third, outer longitudinal muscle layer in the inner sheath of muscles. Mouth opening located at the hind end of the pharyngeal pocket. Numerous ciliated protozoan parasites were present in the intestinal tract of most of the specimens examined (seven out of eight).

Ŋe anterior portion of the oviducts is expanded into a tuba, which communicates with the dorsal part of the ovaries. From thereon, the infranucleated oviducts run ventrally in a caudal direction, immediately dorsal to the ventral nerve cords. Posterior to the genital pore, the oviducts recurve anteriad to open separately and symmetrically into the vaginal portion of the bursal canal ( Fig. 7 ).

Ŋe numerous mature testes are situated dorsally on either side of the body and extend from the level of the ovaries to the posterior end of the body. Vitellaria are located between the testes and the intestinal branches.

Ŋe very large, lobated and sac-shaped copulatory bursa occupies the entire dorso-ventral space; it is lined with a vacuolated, columnar, and glandular epithelium with basal nuclei and is surrounded by a layer of muscles. A spermatophore, filled with sperm, as well as free sperm, were present in the bursa of specimen CGAS Pla 25. 1, together with ciliated parasites ( Fig. 8 ). Ŋese protozoans infested also the lumen of the copulatory bursa in four of the other seven specimens examined. In specimen CGAS Pla 25. 5, parasites were present also in the bursal canal. On the basis of their general morphology and presence of a macronucleus with three micronuclei, these protozoans, infesting both the gut and the copulatory bursa, can be aưributed to the species Haptophrya planariarum (von Siebold, 1839) Stein, 1867 .

From the postero-dorsal wall of the bursa, the bursal canal runs in a caudal direction to the less side of the copulatory apparatus, extending beyond the penis and the genital pore. Posterior to the gonopore, the bursal canal shows a knee-shaped, anteroventrally directed bend, thus forming a so-called ‘angled bursal canal’; hereasser, it opens through the roof of the common genital atrium. Ŋe bursal canal is lined with cylindrical, infranucleated, ciliated cells and is surrounded by a subepithelial layer of longitudinal muscles, followed by a layer of circular muscle. Ectal reinforcement with longitudinal muscles is confined to the posterior tract of the vaginal portion of the bursal canal. Free sperm were present in the bursal canal of specimen CGAS Pla 25. 1. Abundant shell glands discharge their erythrophil secretion into the vaginal section of the bursal canal, just ventrally to the oviducal openings ( Figs 7 , 9A ).

Ŋe well-developed penis bulb consists of intermingled longitudinal and circular muscle fibres. Ŋe penis bulb houses a pleated seminal vesicle, lined with a nucleated epithelium ( Fig. 7 ). Upon reaching the level of the penis bulb, the vasa deferentia curve dorso-medially and then asymmetrically penetrate the wall of the proximal, anterior section of the penis bulb, with the right sperm duct entering antero-dorsally and the less one anteroventrally. Once inside the bulb, the ducts run asymmetrically to reach the posterior portion of the seminal vesicle, close to the diaphragm. Ŋe sperm ducts open asymmetrically into the seminal vesicle, with the right vas deferens opening more dorsally to the less one ( Fig. 7 ). In all specimens examined, the sperm ducts form well-developed spermiducal vesicles, packed with sperm.

Ŋe seminal vesicle opens into the ejaculatory duct via a large, conical diaphragm. Ŋis diaphragm, which is located approximately at the base of the penis papilla, receives the openings of penis glands. Ŋe finger-shaped, symmetrical penis papilla is covered by an infranucleated epithelium, which is underlain with a subepithelial layer of circular muscles, followed by a layer of longitudinal muscle fibres. Ŋe ejaculatory duct follows a central course through the penis papilla and has a terminal opening, while in its anterior tract it receives the openings of erythophil penial glands. Ŋe ejaculatory duct, which in all the specimens examined contained an empty spermatophore, is lined by a cuboidal, infranucleated epithelium ( Figs 7 , 9B ).

A very large penial fold of the parenchymatic-muscular type is situated dorso-laterally or laterally on the less side of the base of the penis papilla. Ŋe fold, with a length that is approximately three-quarters or one-half of that of the penis papilla, is either (i) almost independent of the penis papilla, or (ii) throughout its length aưached to the papilla. Ŋe penial fold is covered by an infranucleated epithelium, which is underlain with a subepithelial layer of circular muscles, followed by a layer of longitudinal muscle fibres ( Figs 7 , 9 ). In specimen CGAS Pla 25. 3, the penial fold shows lateral grooves in its surface. In specimens CGAS Pla 25. 6 ( Fig. 9A ) and RMNH.VER.21055.1 there are also two small muscular atrial folds, which are located ventrally and dorsally, respectively, while a single ventral atrial fold is present in specimen CGAS Pla 25. 2.

Ŋe genital atrium is lined by an infranucleated epithelium, which is underlain by a thick subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibres. Abundant cement glands open into the atrium dorsally to the gonopore. Ŋe atrium opens ventrally through the genital pore ( Fig. 7 ).

Geographical distribution: Sardinia and Capraia Island (Tuscany Archipelago). Originally, it was also considered to be present on Corsica, where it would be the only species of the genus Dugesia inhabiting this Mediterranean island. However, according to our present analysis, its presumed presence on Corsica now has to be evaluated as doubtful.

Reproduction: Sexual and fissiparous. Populations may be exclusively sexual or mixed, the laưer characterized by the coexistence of sexual and fissiparous individuals.

Comparative discussion: In all essential details, the specimens examined from the Su Rizzolu River agree with the features described for D. benazzii by Lepori (1951). However, with respect to diagnostic characters, there are some differences between our observations and those of Lepori (1951). Dugesia benazzii was described with a penial annex or adenodactyl in the form of a muscular-glandular papilla devoid of a lumen that is situated on the less side of the penis papilla and that even may be larger than the penis itself. Moreover, for some specimens from Bunnari and Mascari rivers, Lepori (1951) reported the presence of a muscular-glandular papilla localized ventro-laterally to the penis papilla. Considering that later in the same rivers D. hepta was found, which is characterized by a ventro-lateral fold (see: Stocchino et al., 2005), the description of some specimens could refer precisely to this last-mentioned species instead of nominal D. benazzii . However, more recent studies restricted the term adenodactyl to the strongly muscularized, glandular-parenchymatic folds of the copulatory apparatus that are provided with a lumen ( Stocchino et al., 2017 and references therein). Our analyses revealed that the penial annexes in the specimens examined actually concern a fold, which is shorter than the penis papilla and conforms to the parenchymatic-muscular type sensu Stocchino et al. (2017).

Ŋe original description of D. benazzii does not specify the course of the vasa deferentia or their openings into the seminal vesicle. However, in his horizontal reconstruction drawings of the copulatory apparatus of the Sardinian populations, Lepori (1951: figs 1, 2) depicted a symmetrical course of the vasa deferentia, as well as symmetrical openings into the seminal vesicle. Ŋis contrasts with the situation in our animals, in which the vasa deferentia follow clearly asymmetrical trajectories and also open asymmetrically into the seminal vesicle.

Asymmetrical openings of the vasa deferentia have been reported for two other species of the genus Dugesia viz., D. bifida Stocchino & Sluys, 2014 and D. circumcisa Chen & Dong, 2021 . Dugesia benazzii shares with D. bifida the condition with the right duct opening into the seminal vesicle dorsally to the less one. However, D. benazzii differs from the two above-mentioned species in that its vasa deferentia open close to the diaphragm. In D. bifida , in contrast, the vasa deferentia open halfway into the vesicle, while in D. circumcisa the right sperm duct opens into the anterior portion of the seminal vesicle and the less duct through the mid-lateral wall of the vesicle, with the less sperm duct opening dorsally to the right one ( Stocchino et al., 2014; Wang et al., 2021a).

Cement glands are not mentioned in the original description, while they are well evident in all specimens from the Su Rizzolu River. Interestingly, these glands do not discharge their secretions around the gonopore, as in most of the species of the genus Dugesia , but they open into the atrium ventrally to the opening of the shell glands and dorsally to the genital pore.A similar condition has been reported only for D. subtentaculata (Leria et al.; 2020).

In the original description of D. benazzii, Lepori (1951) reported the presence of a muscular atrial fold only for the Corsican specimens. We also found this feature in our Sardinian specimens. Ŋis atrial annexe has been reported in a few other species of the genus Dugesia viz., D. aenigma de Vries, 1984 , D. ariadnae de Vries, 1984 , D. damoae de Vries, 1984 ( de Vries, 1984; Stocchino et al., 2017), and in D. mariae Stocchino, Dols-Serrate & Riutort , sp. nov. (see below).

Unfortunately, Lepori (1951) did not present any details about the bursal canal. Interestingly, all specimens of the population examined are characterized by an ‘angled’ course of the bursal canal, without exception. It is noteworthy that this character is not common amongst species of Dugesia , whereas it forms a diagnostic character of the genus Girardia ( de Vries and Sluys, 1991; Stocchino et al., 2019).

A short ectal reinforcement confined to the posterior tract of the vaginal portion of the bursal canal is shared with only two species of the genus Dugesia , viz., D. improvisa Sluys & Solà, 2013 and D. naiadis Sluys, 2013 ( Sluys et al., 2013).

In several of our specimens of D. benazzii , the ciliate parasite Haptophryaplanariarum (Phylum Ciliophora) was present in the intestinal tract, the bursal canal, and the copulatory bursa. Specimens of H. planariarum were found also in the gut and copulatory bursa of D. mariae Stocchino, Dols-Serrate & Riutort , sp. nov. (see below), while preliminary observations established the occurrence of these parasites in both the intestinal tract and the copulatory bursa of specimens from other Sardinian populations of D. benazzii , as well as in individuals from some populations of D. hepta (G.A. Stocchino personal observation). It should be noted that the presence of H. planariarum does not seem to affect the development and functionality of the reproductive apparatus of the parasitized individuals.

In addition to these three species of Dugesia , presence of H. planariarum was previously documented for nine other species of freshwater planarian, from which it was generally reported from the gut, the pharynx lumen, or the pharyngeal pouch: Planaria torva ( von Graff, 1903; Bishop, 1926; Lom, 1959; de Puytorac, 1963; Keller, 1983), Schmidtea lugubris ( Lom, 1959; Keller, 1983), Dendrocoelum lacteum ( von Graff, 1903; Bishop, 1926; de Puytorac, 1963), Phagocata iwamai ( Kawakatsu, 1970) , Dugesia gonocephala ( von Graff, 1903; Steinböck, 1924; de Beauchamp, 1932; Gamo Garcia and Rodríguez Caabeiro, 1981;Rataj and Vďačný, 2018), Dugesia monomyoda ( Marcus, 1953) , Polycelis nigra ( von Graff, 1903; de Pyutorac, 1963), Paraplanaria dactyligera ( Kenk, 1969) , Paraplanaria occulta ( Kenk, 1969) . For only D. gonocephala ( Steinböck, 1924; de Beauchamp, 1932) it was reported that H. planariarum parasites occurred also in the copulatory bursa of some specimens. However, recent molecular studies ( Rataj et al., 2022) have highlighted that the morphospecies H. planariarum is a complex of three genetically distinct and morphologically cryptic species. Ŋe other two ciliate species, H. dugesiarum Rataj, Zhang & Vd’ačny, 2022 and H. schmidtearum Rataj, Zhang & Vd’ačny, 2022 , morphologically correspond to H. planariarum and, therefore, these species can be identified only asser molecular analysis. While H. planariarum and H. dugesiarum were found infesting D. gonocephala , H. schmidtearum was found only in Schmidtea polychroa . In all three of the last-mentioned planarian species, the parasites occurred only in the gut and the pharyngeal pouch.

Figures

Figure 1. A, sampling localities of the present study.Numbers correspond to population codes listed in Appendix, Table A1 and coincide with those in Dols-Serrate et al.(2020). Red circles indicate populations used for morphological analyses. B, rectangular inset:enlargement of the Bunnari–Mascari confluence area. Ŋe map was created using Q-GIS v.3.2.2 (hưps://qgis.org/es/site/ last accessed September 2023) and edited in ILLUSTÞTOR CC v.22.0.1 (hưps://www.adobe.com/products/illustrator.html last accessed September 2023).

Figure 6. Karyogram of (A) Dugesia benazzii s.s. from Su Rizzolu River (Oưi, loc. 13) and (B) Dugesia hoidi.

Figure 7. Dugesia benazzii s.s., CGAS Pla 25.1: A, sagiưal reconstruction of the male copulatory apparatus (anterior to the right); B, sagiưal reconstruction of the fold and female copulatory apparatus; C, photomicrograph of sagiưal section, showing the penis bulb (pb), penis papilla (pp) with conical, pointed diaphragm (d), ejaculatory duct (ed), penial fold (pf), and ‘angled’ bursal canal (abc).

Figure 8. Dugesia benazzii s.s., CGAS Pla 25.1. Photomicrograph of the copulatory bursa with a ciliate parasite (cp) and a spermatophore (sp) in a sagiưal section.

Figure 9. Dugesia benazzii s.s.: A, CGAS Pla 25. 6, photomicrograph showing the penial fold (pf) and the two atrial folds (af) in sagiưal section; B, CGAS Pla 25. 4, photomicrograph showing the penis papilla (pp) and the penial fold (pf) in transverse section.