Dugesia mariae Stocchino, Dols&Serrate & Riutort, 2024

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 : 18-21

Dugesia mariae Stocchino, Dols&Serrate & Riutort , sp. nov.

Material examined: Individuals from populations 19, 20, 21, 24, 25, 30, and 31 were analysed molecularly; corresponding GenBank accession numbers are detailed in the Appendix, Table A1 .

Holotype: RMNH.VER.21057.1, tributary of Golo River , locality Barcheưa, (42º30ʹ26.73ʹʹN, 9º04ʹ22.4ʹʹE), Corsica, 17 May 2010, coll. M. Pala, G.A. Stocchino and E.M. Lazaro, one set of sagiưal sections on seven slides.

Paratypes: CGAS Pla 27. 1, ibid., horizontal sections on three slides ; CGAS Pla 27. 2, ibid., horizontal sections onthreeslides ; RMNH.VER.21057.2,ibid., sagittalsectionson eight slides ; CGAS Pla 27. 3p, ibid., sagittal sections on eight slides ; CGAS Pla 27. 4, ibid., transverse sections on 14 slides ; RMNH.VER.21057.3, ibid., horizontal sections on six slides .

Diagnosis: Molecularly, Dugesia mariae comprises individuals that are identified as a single evolutionary unit together with individuals from populations 19, 20, 21, 24, 25, 30, and 31, when using the coalescence-based method BPP, or equivalent methods, with the loci and settings detailed in the present study’s Materials and methods section. Morphologically, D. mariae is characterized by the presence of the following features: short and blunt penis papilla; ventrally displaced ejaculatory duct, opening terminally at tip of the penis papilla; ejaculatory duct broad, with pleated wall and irregularly-shaped lumen; large, conical diaphragm; symmetrical openings of the vasa deferentia into the posterior section of the seminal vesicle, i.e. close to the diaphragm; oviducts opening symmetrically into the vaginal portion of the bursal canal; ectal reinforcement musculature on bursal canal extending from vaginal region almost up to the copulatory bursa; penial fold of the parenchymatic-muscular type, located dorsally or dorso-laterally to the penis papilla; cement glands opening into the genital atrium around the dorsal region of the gonoduct.

Etymology: Ŋe specific epithet refers to Professor emeritus Maria Pala, in recognition of her contributions to the biology and systematics of freshwater triclads.

Geographical distribution: Ŋe species is known from several localities on the island of Corsica: Golo River (Barcheưa, loc. 24; Ponti strada, loc. 25), Fium Alto River (Ecole Communale, loc. 30; San Gavino, loc. 31), Solenzara River (loc. 19), Cioccia River (loc. 20), and Castellu River (Pero Longo, loc. 21).

Reproduction: Sexual and fissiparous. At three localities, only sexual individuals were found (loc. 24, 20, and 21), whereas in three other localities both sexual and fissiparous individuals (loc. 25, 30, and 31) were present, while only fissiparous individuals were found at one locality 19 ( Fig. 1 ).

Description: Ŋe body of preserved specimens measured up to 9.3 mm in length and 2.8 mm in width ( Fig. 12 ). Two eyes are present in the centre of the low triangular head. Dorsal surface brown; ventral surface pale.

Ŋe pharynx is positioned in the mid-region of the body and measures about one-fissh of the body length. Inner and outer pharyngeal musculature bilayered, i.e. without an extra, third, outer longitudinal muscle layer in the inner sheath of muscles. Ŋe mouth opening is situated at the hind end of the pharyngeal pocket. In all specimens examined, the intestinal tract was infested by the ciliate parasite H. planariarum , which was present in both the pharyngeal pouch and the gut.

Ŋe two ovoid ovaries are located ventrally, at a short distance behind the brain, i.e. at about one-third of the distance between the brain and the root of the pharynx. Ŋe anterior portion of the oviducts is expanded into a tuba, which communicates with the dorsal part of the ovaries. From the ovaries, the infranucleated oviducts run ventrally in a caudal direction to the level of the genital pore, whereasser they open separately and symmetrically into the vaginal portion of the bursal canal.

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

Ŋe very large, more or less cuboidal copulatory bursa occupies the entire dorso-ventral space; it is lined by a columnar, vacuolated, and glandular epithelium with basal nuclei and is surrounded by a layer of muscles. Remnants of a spermatophore and sperm are present in the bursa of specimens RMNH. VER.21057.1, CGAS Pla 27. 1, and CGAS Pla 27. 3. From the postero-dorsal wall of the bursa, the bursal canal runs in a caudal direction to the less side of the copulatory apparatus, then curves ventrally just at the level of the gonoduct and opens through the roof of the genital atrium. Ŋe bursal canal is lined with cylindrical, infranucleated, ciliated cells and is surrounded by a thin subepithelial layer of longitudinal muscles, followed by a layer of circular muscle. Ectal reinforcement musculature is present in the form of a well-developed layer of longitudinal muscles running ectally to the circular layer around the bursal canal, extending from the opening of the bursal canal into the atrium to almost the copulatory bursa. Shell glands discharge their erythrophil secretion into the vaginal region of the bursal canal, just ventrally to the oviducal openings ( Fig. 13 ).

Ŋe moderately-developed penis bulb has an oblique, ventrocaudal orientation and consists of intermingled longitudinal and circular muscle fibres. Ŋe penis bulb houses a spacious seminal vesicle, which is lined with a pleated epithelium ( Figs 13 , 14A ).

At the level of the penis bulb, the vasa deferentia curve dorso-medially and then penetrate the anterior wall of the bulb to open separately and symmetrically into the posterior portion of the seminal vesicle, close to the diaphragm. In specimen CGAS Pla 27. 1 the vasa deferentia are swollen to voluminous spermiducal vesicles.

Ŋe seminal vesicle opens into the ejaculatory duct via a well-developed conical diaphragm. Ŋe short and blunt penis papilla is covered by an infranucleated epithelium, which is underlain by a subepithelial layer of circular muscles. Ŋe ejaculatory duct follows a ventral course and has a terminal opening. Ŋe ejaculatory duct, which contained an empty spermatophore in all specimens examined, has an irregularly shaped lumen, due to its considerably pleated wall; it is lined by a cuboidal, infranucleated epithelium and surrounded by a subepithelial layer of longitudinal muscles ( Figs 13 , 14 ).

A penial fold of the parenchymatic-muscular type is situated dorsally or dorso-laterally on the less side of the base of the penis papilla. Ŋe fold measures approximately one-third or one-half of the length of the penis papilla ( Figs 13 , 14B ).

Ŋe genital atrium is lined by a partially infranucleated epithelium, which is underlain by a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibres. Ŋe genital atrium communicates with a gonoduct that is lined by a nucleated columnar epithelium and which opens to the exterior via the ventral gonopore. Cement glands open into the genital atrium around the dorsal region of the gonoduct ( Fig. 13 ). In specimens CGAS Pla 27. 1, RMNH.VER.21057.2, and RMNH. VER.21057.3 there is a small muscular atrial fold that protrudes ventro-laterally as a semicircular valve ( Fig. 14A ).

Comparative discussion: Dugesia mariae differs from its congeners in a unique combination of morphological characters concerning the copulatory apparatus: short and blunt penis papilla with a large, conical diaphragm; ventrally displaced ejaculatory duct, with its terminal opening and its broad lumen and pleated wall; symmetrical openings of the vasa deferentia close to the diaphragm; presence of an ectal reinforcement layer, extending almost up to the copulatory bursa; oviducts opening symmetrically into the vaginal portion of the bursal canal; cement glands opening into the atrium around the dorsal region of a gonoduct; a penial fold of the parenchymatic-muscular type located dorsally or dorso-laterally to the penis papilla.

Both sexual and fissiparous Corsican planarians were first considered by Arndt (1922) as belonging to D. gonocephala . Later, de Beauchamp (1926) assigned the fissiparous populations to D. subtentaculata . Further studies by Benazzi and Lepori led to the description of these populations as a new species, viz., D. benazzii , distributed on both Corsica and Sardinia ( Lepori, 1951 and references therein). However, D. mariae shares with D. benazzii s.s. and D. hoidi only the large, conical diaphragm and the presence of a penial fold on the less side of the penis papilla, whereas it differs from these two species in the asymmetrical penis papilla, the shape of the ejaculatory duct, the extension of the ectal reinforcement, and the absence of an ‘angled’ bursal canal.

With respect to its ventrally displaced ejaculatory duct with terminal opening, D. mariae resembles no less than 28 other species of Dugesia : D. afromontana , D. andamanensis (Kaburaki, 1925) , D. annandalei (Kaburaki, 1918) , D. ariadnae de Vries, 1984 , D. bifida , D. borneana Kawakatsu, 1972 , D. circumcisa Chen & Dong, 2021 , D. damoae de Vries, 1984 , D. deharvengi Kawakatsu & Mitchell, 1989 , D. didiaphragma de Vries, 1988 , D. ectophysa Marcus, 1953 , D. iranica (Livanov, 1951) , D. lanzai Banchetti & Del Papa, 1971 , D. leporii Pala, Stocchino, Corso & Casu, 2000 , D. libanica Bromley & Benazzi, 1991 , D. maghrebiana Stocchino, Manconi, Corso, Sluys, Casu & Pala, 2009 , D. malicky , D. mertoni , D. monomyoda Marcus, 1953 , D. naiadis Sluys, 2013 , D. notogaea Sluys & Kawakatsu, 1998 , D. semiglobosa Chen & Dong, 2021 , D. sinensis Chen & Wang, 2015 , D. pustulata Harrath & Sluys, 2019 , D. parasagitta Sluys & Solà, 2013 , D. ryukyuensis Kawakatsu, 1976 , D. siamana Kawakatsu, 1980 , and D. tubqalis Harrath & Sluys, 2012 .

In seven of these species ( D. andamanensis , D. bifida , D. borneana , D. mertoni , D. notogaea , D. ryukyuensis , and D. siamana ) a duct is present between the seminal vesicle and the diaphragm that is absent in D. mariae . Further, D. mariae shares the presence of a penial fold only with D. iranica , D. libanica , D. malicky , D. parasagita , D. ryukyuensis , D. tubqalis , and Dugesia sp. from Lake Ohrid. However, these species have a small or large—but not conical—diaphragm, in contrast to the large, conical or pointed diaphragm of D. mariae (cf. de Vries, 1984, 1988; Sluys et al., 1998, 2013; Pala et al., 2000; Stocchino et al., 2009, 2012, 2013, 2014, 2017; Harrath et al., 2012, 2019; Chen et al., 2015; Wang et al., 2021a, b).

Ŋe fact that in D. mariae the ectal reinforcement extends to almost the copulatory bursa, contrasts with the situation in D. aþomontana , D. annandalei , D. circumcisa , D. didiaphragma , D. ectophysa , D. iranica , D. maghrebiana , D. monomyoda , D. naiadis , and D. semiglobosa , in which this layer of longitudinal muscles around the bursal canal is completely absent or is confined to the vaginal region of the bursal canal or extends to only to halfway along the canal.

Concurrently, molecular evidence has consistently pointed to the separate specific status of D. mariae throughout all phases of our molecular species delimitation, irrespective of the dataset analysed or the particular methodology employed. Further, it is noteworthy that in a recently published study on the phylotranscriptomics of the genus Dugesia in the Western Mediterranean, evidence was presented for the reciprocal monophyly of the Corsican and Sardinian nominal D. benazzii s.l. populations ( Benítez-Álvarez et al., 2023). For D. hoidi , molecular species delimitation also showed it as a species different from the rest of D. benazzii s.l. populations analysed, a result that is also supported by its differentiated karyotype.

In conclusion, all available evidence, as presented and discussed above, points unequivocally to the separate species status of both D. mariae and D. hoidi .

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 12. Dugesia mariae. Photomicrograph of a preserved sexual specimen from the Golo River (Barcheưa, loc. 24).

Figure 13. Dugesia mariae. Holotype RMNH.VER.21056.1:A, sagiưal reconstruction of the copulatory apparatus (anterior to the right); B, photomicrograph showing, in a sagiưal section, the penis bulb (pb) with seminal vesicle (sv), penis papilla (pp) with the pointed diaphragm (d), pleated ejaculatory duct (ed), and the penial fold (pf).

Figure 14. Dugesia mariae: A, CGAS Pla 27. 1, photomicrograph showing the penis bulb with the seminal vesicle (sv), less (lvd) and right (rvd) vas deferens, the penial papilla (pp) and the two atrial folds (af) in horizontal section; B, CGAS Pla 27. 4, photomicrograph showing the penis papilla (pp) and the penial fold (pf) in transverse section.