A new cotylean polyclad flatworm species from Ghar El Melh lagoon (Northern Tunisia)

A new cotylean polyclad flatworm species from Ghar El Melh lagoon (Northern Tunisia) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A new cotylean polyclad flatworm species from Ghar El Melh lagoon (Northern Tunisia) Mehrez Gammoudi, Isabel Dittmann, Johannes Girstmair, Pavel Tomancak, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3783982/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Nov, 2024 Read the published version in Biologia → Version 1 posted 5 You are reading this latest preprint version Abstract We describe Phrikoceros jannetae sp. nov., a new cotylean polyclad species from Ghar El Melh lagoon, a natural seawater lagoon situated in the north of Tunisia. The new species is characterized by black spots on the dorsal surface in contrast to the white spotted dorsal colour pattern of its congeners. We provide some insights into the biology of this species including the plastic tentacle configuration and the variability of body form and outline within the same specimen. Phrikoceros jannetae sp. nov. was found among tunicates of the species Ciona intestinalis. Western Mediterranean Platyhelminthes morphological variability North African marine biodiversity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Members of Polycladida are almost exclusively marine, free-living flatworms, characterized by a highly branched intestine (Hyman 1951 ; Prudhoe 1985 ). The order Polycladida gather two sub-orders, Acotyela and Cotylea, according to the absence and presence of a sucker behind the female genital pore (Lang 1884 ). Among the Cotylea, the Pseudocerotidae Lang, 1884 is one of the most diverse families, and among them, we can find the most strikingly coloured polyclads. Different authors have considered that their inner anatomy does not provide enough information, due to their seemingly uniform microanatomy, therefore all specific characterization relies mainly on the colour pattern, body outline, tentacle morphology and eye arrangement (Prudhoe 1989 , Newman & Cannon 1995 ). In 2019, Litvaitis and collaborators proposed the suppression of the genus Tytthosoceros and transferred the species gathered here to the genus Phrikoceros , based mostly on molecular data. In 2022, Bahia conducted a short integrative study of these closely related genera and considered this was a possibly premature synonymization and provided molecular as well as morphological evidence supporting the decision for considering Tytthosoceros as a valid genus. In Tunisia, the taxonomy, reproduction, development and ecology of polyclads have been investigated intensively during the last decades (Gammoudi et al. 2009 ; Gammoudi and Tekaya 2012 ; Gammoudi et al. 2012a , 2012b , 2016a , b , c , 2017 ). A considerable number of species has been recorded from lagoonal ecosystems, in particular the lagoon of Tunis, the Bizerta lagoon and the Ghar el Melh lagoon. In this context the polyclad fauna of the Ghar El Melh lagoon, a natural lagoon in the Mediterranean situated in North-eastern Tunisia, on the North-western side of the Gulf of Tunis, has been investigated. The lagoon was designated as a Wetland of International Importance (Ramsar Site) under the Ramsar Convention on 7 November 2007. In this study, we describe a new cotylean species, Phrikoceros jannetae sp. nov., using a combination of external morphology, histology and DNA data. The new species was found among the tunicates Ciona intestinalis (Linnaeus, 1767), suggesting the possibility of a trophic relationship. We aim at testing Bahia’s proposal outlining some external morphological traits as clear diagnostic characters (i.e. tentacle form) for Phrikoceros and Tytthosoceros . Material and methods Specimens were found on the ascidian Ciona intestinalis near the new harbour Marina, Ghar El Melh, Tunisia (37°09'26"N 10°13'18"E) (Fig. 1 ). The animals were hand picked using a fine paintbrush lifting the worms gently off the tunicates. Live specimens were measured in the laboratory. Photographs and morphological study were made using a stereomicroscope Olympus SZX-FOF. Longitudinal sections of the median region of the body were obtained from two individuals fixed with Bouin’s fluid, then dehydrated in a series of increasing concentrations of alcohol. The embedded samples in Paraplast (Sigma-Aldrich, Missouri) were subsequently serially sectioned at 7 µm, and stained with Azan. Photos of histological sections were made using a Leica LCC50E microscope. DNA was extracted from a small piece of tissue and PCR for partial small and large nuclear ribosomal subunits (18S and 28S) was performed following the procedures detailed in Dittmann et al. 2019 , using the primer pair 28S_1F and 28S_6R (Alvarez-Presas et al. 2008). Purified PCR products were sent to Microsynth (Switzerland) for sequencing. Reference sequences of the large ribosomal subunit were downloaded from GenBank and aligned using MAFFT E-INS-i v7.310 (Katoh & Standley 2013 ) with Gblocks v0.91b (Castresana 2000 ) used for alignment curation with least stringent parameters. A maximum likelihood tree reconstruction was calculated with IQ-TREE v2.1.3 (Minh et al. 2020 ), using the integrated ModelFinder to determine the best-fitting model and doing 200 non-parametric bootstrap replicates. The tree file was visualised with FigTree v1.4.3 ( http://tree.bio.ed.ac.uk/ ) Results Taxonomic Account Polycladida Lang, 1881 Cotylea Lang, 1884 Pseudocerotoide a Lang, 1884 Pseudocerotidae Lang, 1884 Phrikoceros Newman & Cannon, 1996 Phrikoceros jannetae sp. nov. Material examined. Holotype: One exemplar collected on September 10th, 2021 in Ghar el Melh lagoon in Tunisia; 37°13'23"N 9°55'57"E. Sagittal sections of the reproductive system were obtained and mounted on 6 slides. Catalogue number: NHMW-ZOO-EV-M-5886 Paratypes: One exemplar was collected on September 10th, 2021 Ghar el Melh lagoon in Tunisia; 37°13'23"N 9°55'57"E. Sagittal sections of the reproductive system were obtained and mounted on 7 slides. Catalogue number: NHMW-ZOO-EV-M-5887 The partial 28S rDNA sequence of the holotype was submitted to GenBank under the accession number OR499798 Etymology. The specific name jannetae is dedicated to the memory of first author´s (MG) beloved mother who passed away in 1999. Description. The body is oval-shaped and fragile, measuring 35 mm long and 19 mm wide at its broadest part (holotype). The second specimen (paratype) measures 30 x 15 mm in length and width. The anterior margin is broad and rounded and only slightly ruffled laterally. Distally it tapers off from the mid-body. The dorsal ground colour is translucent white to brownish, with elongated white blotches that can be arranged in discrete rows and show up as lateral streaks with an irregularly interrupted slender black rim. The dorsal colour pattern consists of randomly distributed black round dots, that become smaller towards the body margin, and red microdots randomly distributed between the black dots (Figs. 2 A-B and 3 C). A mid-dorsal darker band can be discerned, going from the anterior margin, laterally to the brain and reaching its maximum width at the location of the pharynx from where it tapers towards the distal margin. Rhabdites are distributed dorsally throughout the entire dorsal surface (Fig. 4 B). Over the ventral surface, the rhabdites are immersed and richly distributed in the epidermis, especially near the anterior margin and around the gonopores (Figs. 4 A and C). Animals are ventrally translucent and whitish, and show an irregularly interrupted slender black rim visible (Figs. 2 B-C). Dorsal pseudotentacular eyes are arranged in two to three scattered rows (Fig. 3 A). On the ventral side, they are gathered in two dense triangular-shaped clusters near the anterior margin. There, depending on the tentacle´s configuration, they may be organized in four to five lines (Fig. 3 B). For more information on tentacle plasticity, please refer to the discussion section An amply clear area is located dorsally above the brain. In its anterior half a slender black line is centrally situated; the cerebral eyespots are arranged posteriorly as an inverted horseshoes-shaped cluster (Fig. 3 A). Longitudinal sections show that the dorsal body wall consists of an epidermal layer of short-ciliated cylindrical cells with basally situated nuclei. The outlets of the eosinophilic short spindle-shaped rhabdite glands open up between the epidermal cells and are immersed within the parenchyma (Fig. 4 ). Pigment granules are clustered in and below epidermal cells (Fig. 4 A-C). With a thickness of 20 µm, the epidermis is almost four times thicker than the compact and well-developed basement membrane (6 µm). Beneath the basement membrane, the circular muscle layer is almost as thick as the basal membrane (6.4 µm). The 17 µm long longitudinal muscle layer, which blends with the parenchymal transversal muscle bundles, is about two times thicker as the basement membrane. The ventral body wall is weaker in comparison to the dorsal one. The ventral epidermis comprises cylindrical cells alternating with the outlets of numerous rhabdites cells and is about seven times thicker (16 µm) than the much slender basal membrane (2 µm) (Fig. 4 ). Digestive system. The pharynx is located in the anterior third of the body. It is relatively small, narrow, oval-shaped, and extends approximately over 1/6 of the entire body length and shows five pairs of shallow, simple pharyngeal folds (Fig. 2 B-C). The mouth opens in the centre of the pharyngeal cavity. The main intestine stretches over nearly 7/8 of the body length. Starting at the level of the brain, it extends to almost the posterior margin. Lateral branches are arranged dorsally (Fig. 4 C) where they form a dense network of intestinal extensions (anastomosing) reaching the whole margin of the body (Fig. 2 B). Reproductive system. The testes, the small spermiducal bulbs, and the vasa deferentia are arranged ventrally. The male organs are compactly arranged between the posteroventral area of the pharyngeal cavity and the ventral body wall (Fig. 5 B). The male copulatory apparatus is directed forward. The seminal canals lead to the vas deferens which develops small spermiducal bulbs (Fig. 4 C), run posteriorly and laterally to the male copulatory organ up to the level of the vagina. There they turn dorsally and enter the ample seminal vesicle. The seminal vesicle is elongated (300 x 175 µm), oriented posteriorly above the male copulatory organ and also points forward. The ejaculatory duct opens at the distal most part of the vesicle. The first part of the ejaculatory duct develops into spacious cavity, which narrows and continues more ventrally. The inner epithelium of the duct undergoes a transition from squamous to ciliated cuboidal, which is consistent with the duct’s external morphology. The free prostatic vesicle is well developed, measuring half the length of the seminal vesicle, and slightly elongated (140 x 90 µm). Its inner glandular lining is smooth. The intra-vesicular epithelium is responsible for producing all prostatic secretions, as there are no extra-vesicular glands. Both, the seminal and the prostatic vesicle show a slender but apparent muscular wall. For a short distance, the ejaculatory duct and the prostatic duct run parallel inside the penis papilla and join at the base of the sclerotized stylet. The penis papilla is 25 µm long, while the distal, slender, conical, and straight pointing stylet reaches 140 µm. The stylet is fully protected by a penis sheath. Together they are located inside the 170 µm deep male atrium. The epithelium of the penis papilla is non-ciliated. The inner lining of the penis sheath is cuboidal, ciliated and intermingled with glandular cells. Beneath the epithelium, scattered glandular cells are immersed in bundles of muscular fibres, that are oriented parallel to the stylet. The outer epithelium of the sheath and the entire male atrium are coated by a cuboidal to columnar and ciliated epithelium. The male atrium epithelium and the surrounding ventral epidermis exhibit scattered rhabdites. The male gonopore opens 515 µm anterior to the female gonopore, and the ventral sucker is located 1.15 mm posterior to the female gonopore. The ovarian follicles are distributed dorsally throughout the whole body even between intestinal branches and appear in different developmental stages. The uteri are short, not ramified and arranged between the female gonopore and shortly before the sucker. They contain only a few oocytes and are located ventrally to the digestive system and the vasa deferentia. The oviducts enter the vagina posteriorly. The vagina makes a 90-degree ventral turn and continues through a short glandular pouch. This pouch opens to a slender female atrium and the gonopore. The female canal is immersed in a dense mass of cement glands, which mostly open mostly at the glandular pouch. The voluminous glandular mass extends from just behind the seminal vesicle up to 500 µm behind the female pore. Molecular results Our phylogenetic tree (Fig. 6 ) shows Phrikoceros jannetae sp. nov. as sister group to an undetermined Pseudoceros species, both of which are sister group to an undetermined Phrikoceros species. These three sequences are in turn sister group to a clade mostly consisting of Phrikoceros and Monobiceros Faubel, 1984 species. Together, this clade and our new species are the well-supported sister group to a large clade containing Pseudobiceros Faubel, 1984 ; Thysanozoon Grube, 1840; Yungi a Lang, 1884 and Maiazoon Newman & Cannon, 1996 (Fig. 6 ). Biological notes: The marginal pseudotentacles of our new species P. jannetae sp. nov. are two highly moveable, and anteriorly positioned simple folds and appear pointed in the fixed holotype. During collection we categorized the animal’s movement into four main stages. Firstly, the swimming stage, which begins when the animals separate from the substrate in a single, sudden move. Thereby, the body is elongated and oval, with the pseudotentacles pointed and orientated anteriorly. The body displays no ruffles, and the body margin moves anteriorly via two long distal-lateral waves. Secondly, we observed fast gliding, whereby the animal’s body becomes diamond-shaped, with its margin almost devoid of ruffles and the pseudotentacles are orientated anteriorly, and remain pointed (Fig. 7 A). This fast-gliding movement would fit hunting behaviour or searching for food. Thirdly, the worms can exhibit a gentle sliding behaviour, as if exploring their surroundings. In this case, the body is elongated and oval, tapering distally. The margins display a few shallow ruffles and the pseudotentacles are a single anterior square fold (Fig. 7 B). Finally, during slow crawling, the body is elongated and oval, with the lateral margin almost parallel. The margin displays numerous ruffles, and the pseudotentacles appear as a simple fold, arranged dorsally (Fig. 7 C). Discussion P. jannetae sp. nov. has an oval body shape, with marginal tentacles formed by folds of the body margin. It is also has a true sucker located centrally, and a ruffled pharynx which is positioned slightly anteriorly. A single copulatory apparatus is located behind the pharynx. Partly beneath the pharynx, a forward facing, true prostatic vesicle with freely arranged smooth inner glandular lining and a single female apparatus can be found. These features are characteristic for the family Pseudocerotidae (Faubel, 1984 ). The presence of a) a smooth dorsal surface, b) pseudotentacles with dorsal and ventral eye clusters, c) a single male copulatory organ and a single female reproductive system with one female and one male gonopore as well as a well-developed sclerotized stylet and d) the absence of a dorsal anal pore or ramified sperm ducts, rule out most of the genera gathered in this family: Acanthozoon (Collingwood, 1876), Bulaceros (Newman & Cannon, 1996), Maiazoon (Newman & Cannon, 1996), Monobiceros (Faubel, 1984 ), Nymphozoon (Hyman, 1959), Parapseudoceros (Prudhoe, 1989 ), Pseudobiceros (Faubel, 1984 ), Thysanozoon (Grube, 1840) and Yungia (Lang, 1884 ) (Faubel 1984 , Prudhoe 1989 , Neman & Cannon 1996a b, Bahía 2022). The above-listed characters rule out most of the genera in this family, leaving only three pseudocerotid genera that are not excluded. The genus Pseudoceros Lang, 1884 is characterized by the presence of a well-developed ruffled pharynx with numerous and complex folds (“highly ruffled pharynx”, Bahia 2022 ). The presence of a pharynx with only simple folds in P. jannetae sp.nov. also excludes the affiliation of this new species to the genus Pseudoceros. The validity of the remaining genera, Phrikoceros Newman & Cannon, 1996 and Tytthosoceros Newman & Cannon, 1996 has been recently discussed (Litvaitis et al. 2019 , Bahia 2022 ). Both genera were recovered as non-monophyletic in a phylogenetic reconstruction based on partial 28S rDNA, and the genus Tytthosoceros was suppressed as a junior synonym of Phrikoceros (Litvatitis et al. 2019). Bahia ( 2022 ) re-stablished the genus Tytthosoceros based on pseudotentacle morphology and found that several Phrikoceros species correspond to the Tytthosoceros pseudotentacle phenotype, and consequently revised these Phrikoceros species as Tytthosoceros species. Incidentally, all published Phrikoceros sequence data were thus revised as Tytthosoceros , leading (in the absence of Phrikoceros sequences) to a monophyletic genus Tytthosoceros (Bahia 2022 ). To realistically observe the tentacles, the animal was examined alive (Bolaños et al. 2016 , Bahia 2022 ). Phrikoceros jannetae sp. nov. exhibits an extraordinary plasticity in body form and outline, as well as in the form and orientation of its pseudotentacles. We were unable to follow the clear delimitations of pseudotentacle types proposed by Bahía (2022) (Fig. 7 ), which led to the re-stablishement of the genus Tytthosoceros mainly based on this feature. The variability in body form and tentacles of P. jannetae sp. nov., as well as the combination of morphological traits found in the Phrikoceros species (Table 1 ), do not allow it to sustain the clear-cut morphological differences between Phrikoceros and Tytthosoceros proposed by Bahía (2022). Additionally, without molecular data from the type species Phrikoceros baibaiye , it is impossible to recover the genera Tytthosoceros and Phrikoceros in a molecular phylogenetic reconstruction including our own analysis in Fig. 6 . The gathered information does not provide enough evidence to support Bahía´s hypothesis. Therefore, we decided to follow the more conservative conclusion achieved by Litvaitis et al. ( 2019 ) and re-suppress Tytthosoceros as a junior synonym of Phrikoceros . We also describe our animal as a new species of Phrikoceros . For the reasons mentioned above, we consider the genus Phrikoceros to comprise ten valid species: P. baibaiye Newman & Cannon, 1996; P. diadaleos Newman & Cannon, 1996; P. fritillus Newman & Cannon, 1996; P. galacticus Newman & Cannon, 1996, P. inca (Baeza, Veliz, Pardo, et al., 1997), P. katoi Newman & Cannon, 1996, P. lizardensis (Newman & Cannon, 1996), P. mopsus (Marcus, 1952), P. nocturnus (Newman & Cannon, 1996), and P. jannetae sp. nov. Except for P. nocturnus , which is dorsally plain black, all species display a spotted dorsal colour pattern. Of the remaining species, P. mopsus and P. baibaiye have a translucent body similar to that of P. jannetae sp. nov. The dorsal pattern of P. mopsus does not display any black dots or red microdots as it is the case for P. jannetae sp. nov., and the black marginal rim in P. mopsus is not interrupted like in P. jannetae sp. nov. Phrikoceros baibaiye is characterized by orange brown to rust background colour, with white microdots forming larger irregular white streaks and short line of white spots behind the cerebral eyespot, with an interrupted marginal rim of white dots. Phrikoceros jannetae sp. nov. displays a brownish dorsal background colour, with white and brown blotches, red microdots and an interrupted black marginal rim, different from P. baibaiye . The other Phrikoceros species with dorsal microdots are P. fritillus, P. galacticus; P. katoi and P. lizardensis. . The microdots of P. fritillus are brown and its color pattern also displays two unique orange triangular blotches between the tentacles, absent in P. jannetae . The background colour of P. galacticus is dark brown to olive green with white blotches and white microdots evenly dispersed over the surface, different from P. jannetae sp. nov. with a lighter background colour, dark spots and the evenly distributed dorsal red microdots. The microdots in P. katoi are white, evenly distributed over a bright orange dorsal background, except behind the cerebral eyes, where they are arranged in clusters in two clusters, different from the evenly distributed black microdots of P. jannetae . Phrikoceros lizardensis displays white microdots only on the marginal rim, a dark background colour, without dark spots and with two submarginal bands, different form P. jannetae with the evenly distributed red microdots, a lighter background color and well as a single marginal rim, without submarginal bands. The remaining two species of Prikoceros are P. diadaleos and P. inca . The background color of P. diadaleos is orange brown, with creamy spots, without dark spots, without microdots and with two marginal bands, different from P. jannetae with a lighter background color, whithe and black spots, red microdots and a single interrupted marginal rim. On the other hand, P. inca differs from P. jannetae in the brownish yellow background color, the brown spots, without white spots, the two marginal rims and the narrow brown stripe over the mid dorsal surface. The gathered evidence supports the description of a new species of Phrikoceros , P. jannetae sp. nov., from the Western Mediterranean. Declarations Acknowledgments : This work was supported by the Tunisian Ministry of Higher Education and Scientific Research (Programme d’encouragement des jeunes chercheurs, 19PEJC07-19). I. L. D. is a recipient of a DOC Fellowship of the Austrian Academy of Sciences at the Department of Zoology at Universität Innsbruck Conflict of interest The authors declare no conflict of interest. Ethical approval Not applicable . Consent to participate : Not applicable Consent for publication Not applicable Competing interests : The authors have no competing interests to declare that are relevant to the content of this article. 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Prudhoe S (1985) A monograph on polyclad Turbellaria. Oxford University Press, New York. Prudhoe S (1989) Polyclad turbellarians recorded from African waters. Bulletin of the British Museum, Natural History 55: 47–96. Tanu M B, Mahmud Y, Arakawa O, Takatani T, Kajihara H, Kawatsu K, Hamano Y, Asakawa M, Miyazawa K, Noguchi T (2004) Immunoenzymatic visualization of tetrodotoxin (TTX) in Cephalothrix species (Nemertea: Anopla: Palaeonemertea: Cephalotrichidae) and Planocera reticulata (Platyhelminthes: Turbellaria: Polycladida: Planoceridae). Toxicon 44: 515–520. DOI:10.1016/j.toxicon.2004.06.014 Ueda H, Itoi S, Sugita H (2018) TTX-bearing planocerid flatworm (Platyhelminthes: Acotylea) in the Ryukyu Islands, Japan. Marine Drugs 16 (1): 37. DOI:10.3390/md16010037 Table Table 1 is available in the Supplementary Files section. Supplementary Files Table1.docx Cite Share Download PDF Status: Published Journal Publication published 18 Nov, 2024 Read the published version in Biologia → Version 1 posted Editorial decision: Major revisions 12 May, 2024 Reviewers agreed at journal 08 Feb, 2024 Reviewers invited by journal 08 Feb, 2024 Editor assigned by journal 21 Dec, 2023 First submitted to journal 20 Dec, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3783982","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271968193,"identity":"f7e77a5f-4b9d-4c5f-b5a4-d1ff71f084c8","order_by":0,"name":"Mehrez Gammoudi","email":"","orcid":"","institution":"University of Tunis El Manar: Universite de Tunis El Manar","correspondingAuthor":false,"prefix":"","firstName":"Mehrez","middleName":"","lastName":"Gammoudi","suffix":""},{"id":271968194,"identity":"fcdd0409-2761-423f-9b83-96d1c44ed9a0","order_by":1,"name":"Isabel Dittmann","email":"","orcid":"","institution":"Universität Innsbruck: Universitat Innsbruck","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"","lastName":"Dittmann","suffix":""},{"id":271968195,"identity":"fbec3297-292a-47a7-808d-7bf6a3252444","order_by":2,"name":"Johannes Girstmair","email":"","orcid":"","institution":"Max-Planck-Institute of Molecular Cell Biology and Genetics: Max-Planck-Institut fur molekulare Zellbiologie und Genetik","correspondingAuthor":false,"prefix":"","firstName":"Johannes","middleName":"","lastName":"Girstmair","suffix":""},{"id":271968196,"identity":"1d727389-ee9f-494b-bc56-63b3ce8ddd9d","order_by":3,"name":"Pavel Tomancak","email":"","orcid":"","institution":"Max-Planck-Institute of Molecular Cell Biology and Genetics: Max-Planck-Institut fur molekulare Zellbiologie und Genetik","correspondingAuthor":false,"prefix":"","firstName":"Pavel","middleName":"","lastName":"Tomancak","suffix":""},{"id":271968197,"identity":"123cd8e7-399c-48f2-8d72-46f5ca13fdc6","order_by":4,"name":"Bernhard Egger","email":"","orcid":"","institution":"University of Innsbruck: Universitat Innsbruck","correspondingAuthor":false,"prefix":"","firstName":"Bernhard","middleName":"","lastName":"Egger","suffix":""},{"id":271968198,"identity":"608b608c-0bef-40b4-a456-c75964e08fc5","order_by":5,"name":"Verónica N. Bulnes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIie3PsQrCMBCA4SuFulS7pou+QqUgCmJfJaFQFxFHB0GnOOlcJ1/BSRyVQLtE57jp7iKC4KKmHUWDbg754TgI+QgB0On+MhvgIJcjh+UH628Ilssd/Uw8edP8injpJEFk1Qz8dHtkvQGUSwIbl76K8F2ECI/Ikrc9FifguwKbLlcQN+7UEKEM14QFzLaALATOP/aZzE8ZeQR+nJE7DCUxbyriIDsja2OBJClSwJ7AlvIVx+6GdUJDEvMI2GyKqjN+pA0VsQrbjTjTVuCME/PSuzYrpTRkexV5DckxfgE6nU6ne9cT0ktNae6pqEMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6092-4561","institution":"INBIOSUR, Universidad Nacional del Sur, CONICET","correspondingAuthor":true,"prefix":"","firstName":"Verónica","middleName":"N.","lastName":"Bulnes","suffix":""}],"badges":[],"createdAt":"2023-12-21 00:07:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3783982/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3783982/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11756-024-01818-y","type":"published","date":"2024-11-18T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51000047,"identity":"50cbc1f6-9dd5-43ae-a958-dd3efbf93315","added_by":"auto","created_at":"2024-02-12 13:09:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":241260,"visible":true,"origin":"","legend":"\u003cp\u003eSampling locality.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3783982/v1/6242826e99b2692b373d2b0b.png"},{"id":50999569,"identity":"94cae238-7bd8-4b73-a18d-52daa7e5f102","added_by":"auto","created_at":"2024-02-12 13:01:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1020376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhrikoceros jannetae \u003c/em\u003esp. nov.\u003cem\u003e \u003c/em\u003e\u0026nbsp;Holotype NHMW-ZOO-EV-M-5886. \u003cstrong\u003eA\u003c/strong\u003e dorsal view of living specimen; \u003cstrong\u003eB\u003c/strong\u003e diagram of dorsal (lower right) and ventral (upper left) body; \u003cstrong\u003eC\u003c/strong\u003e ventral view of living specimen. Anterior up. Abbreviations: fg, female gonopore; ib, intestinal branches; mg, male gonopore; mi, main intestine; ph, pharynx; su, sucker; te, pseudotentacles\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3783982/v1/78f3f5859435a4f429eaa1e4.png"},{"id":50999571,"identity":"adebcf86-bb51-4925-b754-888a3989b601","added_by":"auto","created_at":"2024-02-12 13:01:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2769011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhrikoceros jannetae \u003c/em\u003esp. nov.\u003cem\u003e \u003c/em\u003e\u0026nbsp;Holotype NHMW-ZOO-EV-M-5886. \u003cstrong\u003eA\u003c/strong\u003e anterior dorsal body margin; \u003cstrong\u003eB\u003c/strong\u003e anterior ventral body margin; \u003cstrong\u003eC\u003c/strong\u003elateral body margin. Anterior up. Abbreviations: ce, cerebral eyes; dte, dorsal pseudotentacular eyes; vte, ventral pseudotentacular eyes.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3783982/v1/a2b7d17c23c4aa5c1ef9dd62.png"},{"id":50999568,"identity":"c30b01fd-8b34-4fc9-969c-840af6f4eb53","added_by":"auto","created_at":"2024-02-12 13:01:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3093119,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhrikoceros jannetae\u003c/em\u003esp. nov. Holotype NHMW-ZOO-EV-M-5886. Microphotograph of histological sagittal sections. \u003cstrong\u003eA\u003c/strong\u003e anterior body at level of a pseudotentacle; \u003cstrong\u003eB\u003c/strong\u003e section through the epidermis showing rhabdites; \u003cstrong\u003eC\u003c/strong\u003e body section through uterus and spermiducal bulb; \u003cstrong\u003eD\u003c/strong\u003e body section through ovaries. Anterior to the left. Abbreviation: br, brain; ce, cerebral eyes; dte, dorsal pseudotentacular eyes; ib, intestinal branch; ov, ovary; pg, pigment granules; ph, pharynx; rh, rhabdites; sb, spermiducal bulbs; ue, uterine eggs; vte, ventral pseudotentacular eyes.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3783982/v1/2a0f0da49bcfbcd069e0669b.png"},{"id":50999573,"identity":"d7358743-edae-4d0b-8915-06daceba472d","added_by":"auto","created_at":"2024-02-12 13:01:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1558485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhrikoceros jannetae \u003c/em\u003esp.nov.Holotype NHMW-ZOO-EV-M-5886. Reproductive system. \u003cstrong\u003eA\u003c/strong\u003e microphotographs of the serial sections of the reproductive system; \u003cstrong\u003eB\u003c/strong\u003e diagram of the reproductive system. Anterior to the left. Abbreviations: cg, cement glands; cp, cement pouch; dvm, dorso-ventral muscles; e, epidermis; ej, ejaculatory duct; fa, female atrium; fg, female gonopore; ma, male atrium; mg, male gonopore; mi, main intestine; ph, pharynx; phc, pharynx cavity; pp, penis papilla; ps, penis sheath; pv, prostatic vesicle; s, stylet; sv, seminal vesicle; uv, uterine vesicles; v, vagina.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3783982/v1/bb7eb2c9d3217b0d8c157d73.png"},{"id":50999574,"identity":"b9d37d22-c7df-41ec-8906-bd5aa52cb6d3","added_by":"auto","created_at":"2024-02-12 13:01:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":306422,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree reconstruction of Pseudocerotidae and \u003cem\u003ePhrikoceros jannetae\u003c/em\u003esp. nov. (green arrow), rooted with \u003cem\u003eCestoplana\u003c/em\u003e. GenBank accession numbers in brackets. Numbers after nodes display bootstrap support of 200 replicates. \u003cem\u003ePseudoceros\u003c/em\u003e (with 62 terminals), \u003cem\u003eThysanozoon\u003c/em\u003e (with 59 terminals), and \u003cem\u003ePhrikoceros mopsus \u003c/em\u003e(with 8 terminals) branches were collapsed for better presentability.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3783982/v1/016ab6f0094c814740381611.png"},{"id":50999572,"identity":"4dbdd2df-60bc-4353-81d4-173d3f590914","added_by":"auto","created_at":"2024-02-12 13:01:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":793211,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhrikoceros jannetae \u003c/em\u003esp. nov. NHMW-ZOO-EV-M-5887. Body margin and pseudotentacle morphology during sliding on a surface. \u003cstrong\u003eA\u003c/strong\u003e fast gliding; \u003cstrong\u003eB\u003c/strong\u003e exploratory sliding; \u003cstrong\u003eC\u003c/strong\u003e slow crawling. Anterior up. Live images were upscaled.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3783982/v1/8a8b3ae44c512000a12c7d4c.png"},{"id":69834849,"identity":"d0131bbd-3d01-425b-9000-4b84742cdaa7","added_by":"auto","created_at":"2024-11-25 16:09:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9383515,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3783982/v1/445f1833-9202-438b-aa23-7fae6c0788ba.pdf"},{"id":50999567,"identity":"3bda9221-fea3-4133-8582-5f8811d61bff","added_by":"auto","created_at":"2024-02-12 13:01:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":675424,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3783982/v1/ca14b939aa00c94fa5e20c01.docx"}],"financialInterests":"","formattedTitle":"A new cotylean polyclad flatworm species from Ghar El Melh lagoon (Northern Tunisia)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMembers of Polycladida are almost exclusively marine, free-living flatworms, characterized by a highly branched intestine (Hyman \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1951\u003c/span\u003e; Prudhoe \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). The order Polycladida gather two sub-orders, Acotyela and Cotylea, according to the absence and presence of a sucker behind the female genital pore (Lang \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1884\u003c/span\u003e). Among the Cotylea, the Pseudocerotidae Lang, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1884\u003c/span\u003e is one of the most diverse families, and among them, we can find the most strikingly coloured polyclads. Different authors have considered that their inner anatomy does not provide enough information, due to their seemingly uniform microanatomy, therefore all specific characterization relies mainly on the colour pattern, body outline, tentacle morphology and eye arrangement (Prudhoe \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1989\u003c/span\u003e, Newman \u0026amp; Cannon \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). In 2019, Litvaitis and collaborators proposed the suppression of the genus \u003cem\u003eTytthosoceros\u003c/em\u003e and transferred the species gathered here to the genus \u003cem\u003ePhrikoceros\u003c/em\u003e, based mostly on molecular data. In 2022, Bahia conducted a short integrative study of these closely related genera and considered this was a possibly premature synonymization and provided molecular as well as morphological evidence supporting the decision for considering \u003cem\u003eTytthosoceros\u003c/em\u003e as a valid genus. In Tunisia, the taxonomy, reproduction, development and ecology of polyclads have been investigated intensively during the last decades (Gammoudi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gammoudi and Tekaya \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Gammoudi et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012b\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003eb\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003ec\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A considerable number of species has been recorded from lagoonal ecosystems, in particular the lagoon of Tunis, the Bizerta lagoon and the Ghar el Melh lagoon. In this context the polyclad fauna of the Ghar El Melh lagoon, a natural lagoon in the Mediterranean situated in North-eastern Tunisia, on the North-western side of the Gulf of Tunis, has been investigated. The lagoon was designated as a Wetland of International Importance (Ramsar Site) under the Ramsar Convention on 7 November 2007. In this study, we describe a new cotylean species, \u003cem\u003ePhrikoceros jannetae\u003c/em\u003e sp. nov., using a combination of external morphology, histology and DNA data. The new species was found among the tunicates \u003cem\u003eCiona intestinalis\u003c/em\u003e (Linnaeus, 1767), suggesting the possibility of a trophic relationship. We aim at testing Bahia\u0026rsquo;s proposal outlining some external morphological traits as clear diagnostic characters (i.e. tentacle form) for \u003cem\u003ePhrikoceros\u003c/em\u003e and \u003cem\u003eTytthosoceros\u003c/em\u003e.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eSpecimens were found on the ascidian \u003cem\u003eCiona intestinalis\u003c/em\u003e near the new harbour Marina, Ghar El Melh, Tunisia (37\u0026deg;09'26\"N 10\u0026deg;13'18\"E) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The animals were hand picked using a fine paintbrush lifting the worms gently off the tunicates. Live specimens were measured in the laboratory. Photographs and morphological study were made using a stereomicroscope Olympus SZX-FOF. Longitudinal sections of the median region of the body were obtained from two individuals fixed with Bouin\u0026rsquo;s fluid, then dehydrated in a series of increasing concentrations of alcohol. The embedded samples in Paraplast (Sigma-Aldrich, Missouri) were subsequently serially sectioned at 7 \u0026micro;m, and stained with Azan. Photos of histological sections were made using a Leica LCC50E microscope. DNA was extracted from a small piece of tissue and PCR for partial small and large nuclear ribosomal subunits (18S and 28S) was performed following the procedures detailed in Dittmann et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, using the primer pair 28S_1F and 28S_6R (Alvarez-Presas et al. 2008). Purified PCR products were sent to Microsynth (Switzerland) for sequencing. Reference sequences of the large ribosomal subunit were downloaded from GenBank and aligned using MAFFT E-INS-i v7.310 (Katoh \u0026amp; Standley \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) with Gblocks v0.91b (Castresana \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) used for alignment curation with least stringent parameters. A maximum likelihood tree reconstruction was calculated with IQ-TREE v2.1.3 (Minh et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), using the integrated ModelFinder to determine the best-fitting model and doing 200 non-parametric bootstrap replicates. The tree file was visualised with FigTree v1.4.3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tree.bio.ed.ac.uk/\u003c/span\u003e\u003cspan address=\"http://tree.bio.ed.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTaxonomic Account\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003ePolycladida Lang, 1881\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section4\"\u003e \u003ch2\u003eCotylea Lang, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1884\u003c/span\u003e\u003c/h2\u003e \u003cp\u003e \u003cb\u003ePseudocerotoide\u003c/b\u003ea Lang, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1884\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePseudocerotidae Lang, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1884\u003c/span\u003e\u003c/h2\u003e \u003cp\u003e \u003cb\u003ePhrikoceros\u003c/b\u003e \u003cb\u003eNewman \u0026amp; Cannon, 1996\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhrikoceros jannetae\u003c/b\u003e \u003cb\u003esp. nov.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eMaterial examined.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHolotype: One exemplar collected on September 10th, 2021 in Ghar el Melh lagoon in Tunisia; 37\u0026deg;13'23\"N 9\u0026deg;55'57\"E. Sagittal sections of the reproductive system were obtained and mounted on 6 slides. Catalogue number: NHMW-ZOO-EV-M-5886\u003c/p\u003e \u003cp\u003eParatypes: One exemplar was collected on September 10th, 2021 Ghar el Melh lagoon in Tunisia; 37\u0026deg;13'23\"N 9\u0026deg;55'57\"E. Sagittal sections of the reproductive system were obtained and mounted on 7 slides. Catalogue number: NHMW-ZOO-EV-M-5887\u003c/p\u003e \u003cp\u003eThe partial 28S rDNA sequence of the holotype was submitted to GenBank under the accession number OR499798\u003c/p\u003e \u003cp\u003e \u003cb\u003eEtymology.\u003c/b\u003e The specific name \u003cem\u003ejannetae\u003c/em\u003e is dedicated to the memory of first author\u0026acute;s (MG) beloved mother who passed away in 1999.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDescription.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe body is oval-shaped and fragile, measuring 35 mm long and 19 mm wide at its broadest part (holotype). The second specimen (paratype) measures 30 x 15 mm in length and width.\u003c/p\u003e \u003cp\u003eThe anterior margin is broad and rounded and only slightly ruffled laterally. Distally it tapers off from the mid-body. The dorsal ground colour is translucent white to brownish, with elongated white blotches that can be arranged in discrete rows and show up as lateral streaks with an irregularly interrupted slender black rim. The dorsal colour pattern consists of randomly distributed black round dots, that become smaller towards the body margin, and red microdots randomly distributed between the black dots (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). A mid-dorsal darker band can be discerned, going from the anterior margin, laterally to the brain and reaching its maximum width at the location of the pharynx from where it tapers towards the distal margin. Rhabdites are distributed dorsally throughout the entire dorsal surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Over the ventral surface, the rhabdites are immersed and richly distributed in the epidermis, especially near the anterior margin and around the gonopores (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and C). Animals are ventrally translucent and whitish, and show an irregularly interrupted slender black rim visible (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C).\u003c/p\u003e \u003cp\u003eDorsal pseudotentacular eyes are arranged in two to three scattered rows (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). On the ventral side, they are gathered in two dense triangular-shaped clusters near the anterior margin. There, depending on the tentacle\u0026acute;s configuration, they may be organized in four to five lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). For more information on tentacle plasticity, please refer to the \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003ediscussion\u003c/span\u003e section\u003c/p\u003e \u003cp\u003eAn amply clear area is located dorsally above the brain. In its anterior half a slender black line is centrally situated; the cerebral eyespots are arranged posteriorly as an inverted horseshoes-shaped cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Longitudinal sections show that the dorsal body wall consists of an epidermal layer of short-ciliated cylindrical cells with basally situated nuclei. The outlets of the eosinophilic short spindle-shaped rhabdite glands open up between the epidermal cells and are immersed within the parenchyma (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Pigment granules are clustered in and below epidermal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). With a thickness of 20 \u0026micro;m, the epidermis is almost four times thicker than the compact and well-developed basement membrane (6 \u0026micro;m). Beneath the basement membrane, the circular muscle layer is almost as thick as the basal membrane (6.4 \u0026micro;m). The 17 \u0026micro;m long longitudinal muscle layer, which blends with the parenchymal transversal muscle bundles, is about two times thicker as the basement membrane. The ventral body wall is weaker in comparison to the dorsal one. The ventral epidermis comprises cylindrical cells alternating with the outlets of numerous rhabdites cells and is about seven times thicker (16 \u0026micro;m) than the much slender basal membrane (2 \u0026micro;m) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDigestive system.\u003c/b\u003e The pharynx is located in the anterior third of the body. It is relatively small, narrow, oval-shaped, and extends approximately over 1/6 of the entire body length and shows five pairs of shallow, simple pharyngeal folds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C).\u003c/p\u003e \u003cp\u003eThe mouth opens in the centre of the pharyngeal cavity. The main intestine stretches over nearly 7/8 of the body length. Starting at the level of the brain, it extends to almost the posterior margin. Lateral branches are arranged dorsally (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) where they form a dense network of intestinal extensions (anastomosing) reaching the whole margin of the body (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eReproductive system.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe testes, the small spermiducal bulbs, and the vasa deferentia are arranged ventrally. The male organs are compactly arranged between the posteroventral area of the pharyngeal cavity and the ventral body wall (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The male copulatory apparatus is directed forward. The seminal canals lead to the vas deferens which develops small spermiducal bulbs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), run posteriorly and laterally to the male copulatory organ up to the level of the vagina. There they turn dorsally and enter the ample seminal vesicle. The seminal vesicle is elongated (300 x 175 \u0026micro;m), oriented posteriorly above the male copulatory organ and also points forward. The ejaculatory duct opens at the distal most part of the vesicle. The first part of the ejaculatory duct develops into spacious cavity, which narrows and continues more ventrally. The inner epithelium of the duct undergoes a transition from squamous to ciliated cuboidal, which is consistent with the duct\u0026rsquo;s external morphology. The free prostatic vesicle is well developed, measuring half the length of the seminal vesicle, and slightly elongated (140 x 90 \u0026micro;m). Its inner glandular lining is smooth. The intra-vesicular epithelium is responsible for producing all prostatic secretions, as there are no extra-vesicular glands. Both, the seminal and the prostatic vesicle show a slender but apparent muscular wall. For a short distance, the ejaculatory duct and the prostatic duct run parallel inside the penis papilla and join at the base of the sclerotized stylet. The penis papilla is 25 \u0026micro;m long, while the distal, slender, conical, and straight pointing stylet reaches 140 \u0026micro;m. The stylet is fully protected by a penis sheath. Together they are located inside the 170 \u0026micro;m deep male atrium. The epithelium of the penis papilla is non-ciliated. The inner lining of the penis sheath is cuboidal, ciliated and intermingled with glandular cells. Beneath the epithelium, scattered glandular cells are immersed in bundles of muscular fibres, that are oriented parallel to the stylet. The outer epithelium of the sheath and the entire male atrium are coated by a cuboidal to columnar and ciliated epithelium. The male atrium epithelium and the surrounding ventral epidermis exhibit scattered rhabdites. The male gonopore opens 515 \u0026micro;m anterior to the female gonopore, and the ventral sucker is located 1.15 mm posterior to the female gonopore. The ovarian follicles are distributed dorsally throughout the whole body even between intestinal branches and appear in different developmental stages. The uteri are short, not ramified and arranged between the female gonopore and shortly before the sucker. They contain only a few oocytes and are located ventrally to the digestive system and the vasa deferentia. The oviducts enter the vagina posteriorly. The vagina makes a 90-degree ventral turn and continues through a short glandular pouch. This pouch opens to a slender female atrium and the gonopore. The female canal is immersed in a dense mass of cement glands, which mostly open mostly at the glandular pouch. The voluminous glandular mass extends from just behind the seminal vesicle up to 500 \u0026micro;m behind the female pore.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMolecular results\u003c/h2\u003e \u003cp\u003eOur phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) shows \u003cem\u003ePhrikoceros jannetae\u003c/em\u003e sp. nov. as sister group to an undetermined \u003cem\u003ePseudoceros\u003c/em\u003e species, both of which are sister group to an undetermined \u003cem\u003ePhrikoceros\u003c/em\u003e species. These three sequences are in turn sister group to a clade mostly consisting of \u003cem\u003ePhrikoceros\u003c/em\u003e and \u003cem\u003eMonobiceros\u003c/em\u003e Faubel, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1984\u003c/span\u003e species. Together, this clade and our new species are the well-supported sister group to a large clade containing \u003cem\u003ePseudobiceros\u003c/em\u003e Faubel, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; \u003cem\u003eThysanozoon\u003c/em\u003e Grube, 1840; \u003cem\u003eYungi\u003c/em\u003ea Lang, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1884\u003c/span\u003e and \u003cem\u003eMaiazoon\u003c/em\u003e Newman \u0026amp; Cannon, 1996 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBiological notes:\u003c/h2\u003e \u003cp\u003eThe marginal pseudotentacles of our new species \u003cem\u003eP. jannetae\u003c/em\u003e sp. nov. are two highly moveable, and anteriorly positioned simple folds and appear pointed in the fixed holotype. During collection we categorized the animal\u0026rsquo;s movement into four main stages. Firstly, the swimming stage, which begins when the animals separate from the substrate in a single, sudden move. Thereby, the body is elongated and oval, with the pseudotentacles pointed and orientated anteriorly. The body displays no ruffles, and the body margin moves anteriorly via two long distal-lateral waves. Secondly, we observed fast gliding, whereby the animal\u0026rsquo;s body becomes diamond-shaped, with its margin almost devoid of ruffles and the pseudotentacles are orientated anteriorly, and remain pointed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). This fast-gliding movement would fit hunting behaviour or searching for food. Thirdly, the worms can exhibit a gentle sliding behaviour, as if exploring their surroundings. In this case, the body is elongated and oval, tapering distally. The margins display a few shallow ruffles and the pseudotentacles are a single anterior square fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Finally, during slow crawling, the body is elongated and oval, with the lateral margin almost parallel. The margin displays numerous ruffles, and the pseudotentacles appear as a simple fold, arranged dorsally (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eP. jannetae\u003c/em\u003e sp. nov. has an oval body shape, with marginal tentacles formed by folds of the body margin. It is also has a true sucker located centrally, and a ruffled pharynx which is positioned slightly anteriorly. A single copulatory apparatus is located behind the pharynx. Partly beneath the pharynx, a forward facing, true prostatic vesicle with freely arranged smooth inner glandular lining and a single female apparatus can be found. These features are characteristic for the family Pseudocerotidae (Faubel, \u003cspan class=\"CitationRef\"\u003e1984\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe presence of a) a smooth dorsal surface, b) pseudotentacles with dorsal and ventral eye clusters, c) a single male copulatory organ and a single female reproductive system with one female and one male gonopore as well as a well-developed sclerotized stylet and d) the absence of a dorsal anal pore or ramified sperm ducts, rule out most of the genera gathered in this family: \u003cem\u003eAcanthozoon\u003c/em\u003e (Collingwood, 1876), \u003cem\u003eBulaceros\u003c/em\u003e (Newman \u0026amp; Cannon, 1996), \u003cem\u003eMaiazoon\u003c/em\u003e (Newman \u0026amp; Cannon, 1996), \u003cem\u003eMonobiceros\u003c/em\u003e (Faubel, \u003cspan class=\"CitationRef\"\u003e1984\u003c/span\u003e), \u003cem\u003eNymphozoon\u003c/em\u003e (Hyman, 1959), \u003cem\u003eParapseudoceros\u003c/em\u003e (Prudhoe, \u003cspan class=\"CitationRef\"\u003e1989\u003c/span\u003e), \u003cem\u003ePseudobiceros\u003c/em\u003e (Faubel, \u003cspan class=\"CitationRef\"\u003e1984\u003c/span\u003e), \u003cem\u003eThysanozoon\u003c/em\u003e (Grube, 1840) and \u003cem\u003eYungia\u003c/em\u003e (Lang, \u003cspan class=\"CitationRef\"\u003e1884\u003c/span\u003e) (Faubel \u003cspan class=\"CitationRef\"\u003e1984\u003c/span\u003e, Prudhoe \u003cspan class=\"CitationRef\"\u003e1989\u003c/span\u003e, Neman \u0026amp; Cannon 1996a b, Bah\u0026iacute;a 2022). The above-listed characters rule out most of the genera in this family, leaving only three pseudocerotid genera that are not excluded. The genus \u003cem\u003ePseudoceros\u003c/em\u003e Lang, \u003cspan class=\"CitationRef\"\u003e1884\u003c/span\u003e is characterized by the presence of a well-developed ruffled pharynx with numerous and complex folds (\u0026ldquo;highly ruffled pharynx\u0026rdquo;, Bahia \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). The presence of a pharynx with only simple folds in \u003cem\u003eP. jannetae\u003c/em\u003e sp.nov. also excludes the affiliation of this new species to the genus \u003cem\u003ePseudoceros.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe validity of the remaining genera, \u003cem\u003ePhrikoceros\u003c/em\u003e Newman \u0026amp; Cannon, 1996 and \u003cem\u003eTytthosoceros\u003c/em\u003e Newman \u0026amp; Cannon, 1996 has been recently discussed (Litvaitis et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e, Bahia \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Both genera were recovered as non-monophyletic in a phylogenetic reconstruction based on partial 28S rDNA, and the genus \u003cem\u003eTytthosoceros\u003c/em\u003e was suppressed as a junior synonym of \u003cem\u003ePhrikoceros\u003c/em\u003e (Litvatitis et al. 2019). Bahia (\u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e) re-stablished the genus \u003cem\u003eTytthosoceros\u003c/em\u003e based on pseudotentacle morphology and found that several \u003cem\u003ePhrikoceros\u003c/em\u003e species correspond to the \u003cem\u003eTytthosoceros\u003c/em\u003e pseudotentacle phenotype, and consequently revised these \u003cem\u003ePhrikoceros\u003c/em\u003e species as \u003cem\u003eTytthosoceros\u003c/em\u003e species. Incidentally, all published \u003cem\u003ePhrikoceros\u003c/em\u003e sequence data were thus revised as \u003cem\u003eTytthosoceros\u003c/em\u003e, leading (in the absence of \u003cem\u003ePhrikoceros\u003c/em\u003e sequences) to a monophyletic genus \u003cem\u003eTytthosoceros\u003c/em\u003e (Bahia \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eTo realistically observe the tentacles, the animal was examined alive (Bola\u0026ntilde;os et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e, Bahia \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003ePhrikoceros jannetae\u003c/em\u003e sp. nov. exhibits an extraordinary plasticity in body form and outline, as well as in the form and orientation of its pseudotentacles. We were unable to follow the clear delimitations of pseudotentacle types proposed by Bah\u0026iacute;a (2022) (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e), which led to the re-stablishement of the genus \u003cem\u003eTytthosoceros\u003c/em\u003e mainly based on this feature.\u003c/p\u003e\n\u003cp\u003eThe variability in body form and tentacles of \u003cem\u003eP. jannetae\u003c/em\u003e sp. nov., as well as the combination of morphological traits found in the \u003cem\u003ePhrikoceros\u003c/em\u003e species (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), do not allow it to sustain the clear-cut morphological differences between \u003cem\u003ePhrikoceros\u003c/em\u003e and \u003cem\u003eTytthosoceros\u003c/em\u003e proposed by Bah\u0026iacute;a (2022).\u003c/p\u003e\n\u003cp\u003eAdditionally, without molecular data from the type species \u003cem\u003ePhrikoceros baibaiye\u003c/em\u003e, it is impossible to recover the genera \u003cem\u003eTytthosoceros\u003c/em\u003e and \u003cem\u003ePhrikoceros\u003c/em\u003e in a molecular phylogenetic reconstruction including our own analysis in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eThe gathered information does not provide enough evidence to support Bah\u0026iacute;a\u0026acute;s hypothesis. Therefore, we decided to follow the more conservative conclusion achieved by Litvaitis et al. (\u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e) and re-suppress \u003cem\u003eTytthosoceros\u003c/em\u003e as a junior synonym of \u003cem\u003ePhrikoceros\u003c/em\u003e. We also describe our animal as a new species of \u003cem\u003ePhrikoceros\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFor the reasons mentioned above, we consider the genus \u003cem\u003ePhrikoceros\u003c/em\u003e to comprise ten valid species: \u003cem\u003eP. baibaiye\u003c/em\u003e Newman \u0026amp; Cannon, 1996; \u003cem\u003eP. diadaleos\u003c/em\u003e Newman \u0026amp; Cannon, 1996; \u003cem\u003eP. fritillus\u003c/em\u003e Newman \u0026amp; Cannon, 1996; \u003cem\u003eP. galacticus\u003c/em\u003e Newman \u0026amp; Cannon, 1996, \u003cem\u003eP. inca\u003c/em\u003e (Baeza, Veliz, Pardo, et al., 1997), \u003cem\u003eP. katoi\u003c/em\u003e Newman \u0026amp; Cannon, 1996, \u003cem\u003eP. lizardensis\u003c/em\u003e (Newman \u0026amp; Cannon, 1996), \u003cem\u003eP. mopsus\u003c/em\u003e (Marcus, 1952), \u003cem\u003eP. nocturnus\u003c/em\u003e (Newman \u0026amp; Cannon, 1996), and \u003cem\u003eP. jannetae\u003c/em\u003e sp. nov. Except for \u003cem\u003eP. nocturnus\u003c/em\u003e, which is dorsally plain black, all species display a spotted dorsal colour pattern. Of the remaining species, \u003cem\u003eP. mopsus and P. baibaiye\u003c/em\u003e have a translucent body similar to that of \u003cem\u003eP. jannetae\u003c/em\u003e sp. nov. The dorsal pattern of \u003cem\u003eP. mopsus\u003c/em\u003e does not display any black dots or red microdots as it is the case for \u003cem\u003eP. jannetae\u003c/em\u003e sp. nov., and the black marginal rim in \u003cem\u003eP. mopsus\u003c/em\u003e is not interrupted like in \u003cem\u003eP. jannetae\u003c/em\u003e sp. nov. \u003cem\u003ePhrikoceros baibaiye\u003c/em\u003e is characterized by orange brown to rust background colour, with white microdots forming larger irregular white streaks and short line of white spots behind the cerebral eyespot, with an interrupted marginal rim of white dots. \u003cem\u003ePhrikoceros jannetae\u003c/em\u003e sp. nov. displays a brownish dorsal background colour, with white and brown blotches, red microdots and an interrupted black marginal rim, different from \u003cem\u003eP. baibaiye\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe other \u003cem\u003ePhrikoceros\u003c/em\u003e species with dorsal microdots are \u003cem\u003eP. fritillus, P. galacticus; P. katoi and P. lizardensis.\u003c/em\u003e. The microdots of \u003cem\u003eP. fritillus\u003c/em\u003e are brown and its color pattern also displays two unique orange triangular blotches between the tentacles, absent in \u003cem\u003eP. jannetae\u003c/em\u003e. The background colour of \u003cem\u003eP. galacticus\u003c/em\u003e is dark brown to olive green with white blotches and white microdots evenly dispersed over the surface, different from \u003cem\u003eP. jannetae\u003c/em\u003e sp. nov. with a lighter background colour, dark spots and the evenly distributed dorsal red microdots. The microdots in \u003cem\u003eP. katoi\u003c/em\u003e are white, evenly distributed over a bright orange dorsal background, except behind the cerebral eyes, where they are arranged in clusters in two clusters, different from the evenly distributed black microdots of \u003cem\u003eP. jannetae\u003c/em\u003e. \u003cem\u003ePhrikoceros lizardensis\u003c/em\u003e displays white microdots only on the marginal rim, a dark background colour, without dark spots and with two submarginal bands, different form P. jannetae with the evenly distributed red microdots, a lighter background color and well as a single marginal rim, without submarginal bands. The remaining two species of \u003cem\u003ePrikoceros\u003c/em\u003e are \u003cem\u003eP. diadaleos\u003c/em\u003e and \u003cem\u003eP. inca\u003c/em\u003e. The background color of \u003cem\u003eP. diadaleos\u003c/em\u003e is orange brown, with creamy spots, without dark spots, without microdots and with two marginal bands, different from \u003cem\u003eP. jannetae\u003c/em\u003e with a lighter background color, whithe and black spots, red microdots and a single interrupted marginal rim. On the other hand, \u003cem\u003eP. inca\u003c/em\u003e differs from \u003cem\u003eP. jannetae\u003c/em\u003e in the brownish yellow background color, the brown spots, without white spots, the two marginal rims and the narrow brown stripe over the mid dorsal surface.\u003c/p\u003e\n\u003cp\u003eThe gathered evidence supports the description of a new species of \u003cem\u003ePhrikoceros\u003c/em\u003e, \u003cem\u003eP. jannetae\u003c/em\u003e sp. nov., from the Western Mediterranean.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e: This work was supported by the Tunisian Ministry of Higher Education and Scientific Research \u0026nbsp;(Programme d\u0026rsquo;encouragement des jeunes chercheurs, 19PEJC07-19). I. L. D. is a recipient of a DOC Fellowship of the Austrian Academy of Sciences at the Department of Zoology at Universit\u0026auml;t Innsbruck\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e Not applicable .\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors have no competing interests to declare that are relevant to the content of this article.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u0026Aacute;lvarez-Presas M, Bagu\u0026ntilde;\u0026agrave; J, Riutort M (2008) Molecular phylogeny of land and freshwater planarians (Tricladida, Platyhelminthes): From freshwater to land and back. Molecular Phylogenetics and Evolution 47(2): 555\u0026ndash;568. DOI: 10.1016/j.ympev.2008.01.032.\u003c/li\u003e\n \u003cli\u003eBaeza J A, V\u0026eacute;liz D, Pardo L M, Lohrmann K B, Guisado C A (1997) A New Polyclad Flatworm, \u003cem\u003eTytthosoceros inca\u003c/em\u003e (Plathyhelminthes: Polycladida: Cotylea: Pseudocerotidae), From Chilean Coastal Waters. Proceedings of the biological society of Washington 110 (3):476\u0026ndash;482\u003c/li\u003e\n \u003cli\u003eBahia J (2022) Twofold or threefold complication: \u003cem\u003ePhrikoceros\u003c/em\u003e or \u003cem\u003eTytthosoceros\u0026nbsp;\u003c/em\u003e(Platyhelminthes: Pseudocerotidae) in South America? Short integrative revision of both genera. Zootaxa 5205 (5): 496\u0026ndash;50. 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DOI:10.1111/are.12995\u003c/li\u003e\n \u003cli\u003eGammoudi M, Salvenmoser W, Tekaya S, Egger B (2016c) Ultrastructure of the ovary and oogenesis in the flatworm \u003cem\u003eProsthiostomum siphunculus\u003c/em\u003e (Polycladida, Cotylea). Cell Biology International40 (11): 1174\u0026ndash;1186. DOI:10.1002/cbin.10657\u003c/li\u003e\n \u003cli\u003eGammoudi M, Garbouj M, Egger B \u0026amp; Tekaya S (2017) Updated inventory and distribution of free-living flatworms from Tunisian waters. Zootaxa 4263 (1): 120\u0026ndash;138. DOI:10.11646/zootaxa.4263.1.5\u003c/li\u003e\n \u003cli\u003eHyman L H (1951) The invertebrates: Platyhelminthes and Rhynchocoela. The acoelomate Bilateria. Vol. II. McGraw-Hill, New York.\u003c/li\u003e\n \u003cli\u003eKatoh K, Standley D M (2013) MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution 30: 772\u0026ndash;780. DOI:10.1093/molbev/mst010\u003c/li\u003e\n \u003cli\u003eLang A (1884) Die Polycladen (Seeplanarien) des Golfes von Neapel und der angrenzenden Meeresabschnitte. Eine Monographie. Fauna und Flora des Golfes von Neapel und der angrenzenden Meeresabschnitte, herausgegeben von der Zoologische Station in Neapel, Engelmann, Leipzig.\u003c/li\u003e\n \u003cli\u003eLitvaitis M K, Bola\u0026ntilde;os D M, Quiroga S Y (2019) Systematic congruence in Polycladida (Platyhelminthes, Rhabditophora): are DNA and morphology telling the same story? Zoological Journal of the Linnean Society186: 865\u0026ndash;891. DOI:10.1093/zoolinnean/zlz007\u003c/li\u003e\n \u003cli\u003eMcNab J M, Rodr\u0026iacute;guez J, Karuso P, Williamson J E (2021) Natural Products in Polyclad Flatworms. Marine drugs 19 (2): 47. DOI:10.3390/md19020047\u003c/li\u003e\n \u003cli\u003eMinh B Q, Hahn M W, Lanfear R (2020) New methods to calculate concordance factors for phylogenomic datasets. 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Oxford University Press, New York.\u003c/li\u003e\n \u003cli\u003ePrudhoe S (1989) Polyclad turbellarians recorded from African waters. Bulletin of the British Museum, Natural History 55: 47\u0026ndash;96.\u003c/li\u003e\n \u003cli\u003eTanu M B, Mahmud Y, Arakawa O, Takatani T, Kajihara H, Kawatsu K, Hamano Y, Asakawa M, Miyazawa K, Noguchi T (2004) Immunoenzymatic visualization of tetrodotoxin (TTX) in \u003cem\u003eCephalothrix\u003c/em\u003e species (Nemertea: Anopla: Palaeonemertea: Cephalotrichidae) and \u003cem\u003ePlanocera reticulata\u003c/em\u003e (Platyhelminthes: Turbellaria: Polycladida: Planoceridae). Toxicon 44: 515\u0026ndash;520. DOI:10.1016/j.toxicon.2004.06.014\u003c/li\u003e\n \u003cli\u003eUeda H, Itoi S, Sugita H (2018) TTX-bearing planocerid flatworm (Platyhelminthes: Acotylea) in the Ryukyu Islands, Japan. Marine Drugs 16 (1): 37. DOI:10.3390/md16010037\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Western Mediterranean, Platyhelminthes, morphological variability, North African marine biodiversity","lastPublishedDoi":"10.21203/rs.3.rs-3783982/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3783982/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe describe \u003cem\u003ePhrikoceros jannetae\u003c/em\u003e sp. nov., a new cotylean polyclad species from Ghar El Melh lagoon, a natural seawater lagoon situated in the north of Tunisia. The new species is characterized by black spots on the dorsal surface in contrast to the white spotted dorsal colour pattern of its congeners. We provide some insights into the biology of this species including the plastic tentacle configuration and the variability of body form and outline within the same specimen. \u003cem\u003ePhrikoceros jannetae\u003c/em\u003e sp. nov. was found among tunicates of the species \u003cem\u003eCiona intestinalis.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"A new cotylean polyclad flatworm species from Ghar El Melh lagoon (Northern Tunisia)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-12 13:01:46","doi":"10.21203/rs.3.rs-3783982/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-05-12T10:21:22+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-02-08T08:38:32+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-08T08:29:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-22T01:44:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biologia","date":"2023-12-20T12:04:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"18bb03f6-13c5-4f94-9044-12109beecfa9","owner":[],"postedDate":"February 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-25T16:01:12+00:00","versionOfRecord":{"articleIdentity":"rs-3783982","link":"https://doi.org/10.1007/s11756-024-01818-y","journal":{"identity":"biologia","isVorOnly":false,"title":"Biologia"},"publishedOn":"2024-11-18 15:57:23","publishedOnDateReadable":"November 18th, 2024"},"versionCreatedAt":"2024-02-12 13:01:46","video":"","vorDoi":"10.1007/s11756-024-01818-y","vorDoiUrl":"https://doi.org/10.1007/s11756-024-01818-y","workflowStages":[]},"version":"v1","identity":"rs-3783982","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3783982","identity":"rs-3783982","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}