A New Polyclad Flatworm, Idiostylochus tortuosus gen. nov., sp. nov. (Platyhelminthes, Polycladida) from France. Can this Foreign Flatworm be Responsible for the Deterioration of Oyster and Mussel Farms?

© 2023 Academia Sinica, Taiwan Open Access A New Polyclad Flatworm, Idiostylochus tortuosus gen. nov., sp. nov. (Platyhelminthes, Polycladida) from France. Can this Foreign Flatworm be Responsible for the Deterioration of Oyster and Mussel Farms? Adrian Gutiérrez1, Isabelle Auby2, Benoit Gouillieux3, Guillemine Daffe3, Cecile Massé4, Elvire Antajan2, and Carolina Noreña1,* 1Departamento de Biodiversidad y Biologia Evolutiva, Museo Nacional de Ciencias Naturales (CSIC), C/ Jose Gutierrez Abascal 2, 28006 Madrid, Spain. *Correspondence: E-mail: [email protected] (Noreña). E-mail: [email protected] (Gutiérrez) 2Ifremer, LITTORAL, Quai Du Commandant Silhouette, 33120, Arcachon, France. E-mail: [email protected] (Auby); [email protected] (Antajan) 3Univ. Bordeaux, CNRS, Observatoire Aquitain des Sciences de l’Univers, UAR 2567 POREA, F-33615, Pessac, France. E-mail: [email protected] (Gouillieux); [email protected] (Daffe) 4Service Patrimoine Naturel (PATRINAT) OFB, CNRS, MNHN, CP41, 36 rue Geoffroy Saint-Hilaire 75005 Paris, France. E-mail: [email protected] (Masse) Received 19 March 2022 / Accepted 18 January 2023 / Published 26 May 2023 Communicated by Benny K.K. Chan A new species of polyclad flatworm, Idiostylochus tortuosus gen. nov., sp. nov. (Polycladida, Idioplanidae), from Arcachon Bay (France) is described. This description is based on a morphological analysis and a molecular analysis using partial sequences of the 28S and cytochrome Oxidase I (COI) genes. After the molecular analysis Idiostylochus gen. nov. appears to be the second genus of the Family Idioplanidae and closely related to the family Latocestidae as well as the genera Leptostylochus and Mirostylochus. The molecular data revealed that the new species may belong to an Indonesian or Indo-Pacific family, closely related to genera with origins in South Pacific Ocean waters. This species was found feeding on the oysters and mussels of the Arcachon farms. Key words: Acotylea, Idioplanidae, Non-native species, Magallana gigas, Oyster culture. Citation: Gutiérrez A, Auby I, Gouillieux B, Daffe G, Massé C, Antajan E, Noreña C. 2023. A new polyclad flatworm, Idiostylochus tortuosus gen. nov., sp. nov. (Platyhelminthes, Polycladida) from France. Can this foreign flatworm be responsible for the deterioration of oyster and mussel farms? Zool Stud 62:15. doi:10.6620/ZS.2023.62-15. BACKGROUND France is the main producer and consumer of oysters in Europe. French oyster farms produce around 80,000 tons of Japanese oysters (Magallana gigas (Thunberg, 1793), formerly Crassostrea gigas) per year, representing 78% of European annual production (FAO 2021). With an annual production, in normal conditions, of around 10,000 tons of M. gigas (Vieira et al. 2020), Arcachon Bay is not only one of the most important areas for French oyster farming, it is also one of the first areas to have implemented this type of culture (Bouchet et al. 1997; Buestel et al. 2009). Over the years, three different species of oysters have been farmed in Arcachon Bay: the European flat oyster (Ostrea edulis Linnaeus, 1758), the Portuguese cupped oyster (Crassostrea angulata (Lamarck, 1819)), and the abovementioned Japanese oyster. The appearance of different Zoological Studies 62:15 (2023) doi:10.6620/ZS.2023.62-15 1 © 2023 Academia Sinica, Taiwan epizootics caused the collapse of the European and Portuguese species. At present, French oyster farmers focus their cultures almost exclusively on M. gigas. This species was introduced to Arcachon in the 1970s to gradually replace the declining oyster cultures of C. angulata (Buestel et al. 2009). Arcachon Bay benefited in May 1971 from an initial input of 52.5 tonnes of broodstock from British Columbia in Canada (very large oysters about 10 years old), reinforced by 60 tonnes in 1972 and 25 tonnes in 1973, making a total of 137.5 tonnes of mother oysters that were placed in several reserves along the Basin. At the same time, large quantities of spat from Japan were introduced (Grizel and Héral 1991). Magallana gigas is characterized by its large size, rapid growth and high tolerance to environmental changes compared to other oyster species (FAO 2005–2021). Since the introduction of Magallana gigas from Japan, Canada and North America in the 1970s (Grizel and Héral 1991), several non-indigenous species have been observed (e.g., Bachelet et al. 2009; Lavesque et al. 2013; Gouillieux and Massé 2019), introduced directly from the Pacific or during regular transports of batches between French oyster farming areas. The description of a new species in the Arcachon Bay, which ultimately turned out to be a non-indigenous species, occurred recently with the annelid polychaete Marphysa victori Lavesque, Daffe, Bonifácio & Hutchings, 2017 (Annelida) (Lavesque et al. 2020). In 2020, the presence of large amounts of unknown polyclads (Platyhelminthes, Polycladida) feeding on individuals of M. gigas and Mytilus sp. were recorded in Arcachon Bay. The occurrence of this polyclad may be related to the increasing oyster mortality recorded since 2019 in oyster farms (Vieira et al. 2020) and mussel beds in Arcachon Bay (local marine fisheries committee, pers. com.) Most polyclads are predators of other small invertebrates, such as crustaceans, ascidians, cnidarians, gastropods, or bivalves (Barton et al. 2020; Jennings 1957; Newman and Cannon 2003; Lee 2006; Teng et al. 2022). At present, the suborder Acotylea (Polycladida) is divided into three superfamilies: Discoceloidea Dittmann, Cuadrado, Aguado, Noreña and Egger, 2019; Leptoplanoidea Faubel, 1984; and Stylochoidea Poche, 1926. Within Acotylea, known predators of bivalves belong mainly to the superfamily Stylochoidea. They prey on mussels (Galleni et al. 1980), scallops (Heasman et al. 1998), giant clams (Newman et al. 1993) and oysters (Danglade 1919; Pearse and Wharton 1938; Littlewood and Marsbe 1990; Newman et al. 1993). Although the mechanism used to open the valves of the prey varies among species, this process usually starts with the polyclad gliding over the posterior end of the valves, followed by the secretion of a considerable amount of mucus. This secretion could serve to immobilize the prey (Hyman 1951), as well as to avoid desiccation and potential attacks by other predators (Gammoudi et al. 2017). Once in position, the polyclad inserts its pharynx into the bivalve, damages the adductor muscle to prevent the prey from closing its valves, and digests it (Gammoudi et al. 2017). The aim of this study is to describe and determine the systematic position of a new species of Polycladida found in Arcachon Bay. These animals are known as predators of bivalve mollusks, and the molecular evidence suggests that they are a non-native species. These preliminary results will guide future studies focused on the habitat, ecology and impact of this species in France. MATERIALS AND METHODS Sampling The specimens were collected by hand in the Arcachon Bay (French Atlantic Coast) (Fig. 1) in October 2020. The live specimens were anesthetized with a solution of 7% MgCl2 and photographed. A small sample of tissue from the lateral margin was preserved in absolute ethanol for DNA extraction. The rest of the specimens were fixed individually in frozen 10% formalin buffered with filtered seawater. After 24 h, the worms were transferred to 30% ethanol for an hour, then to 50% ethanol for an hour and finally stored in 70% ethanol. Histological processing The fixed specimens were dehydrated in progressive ethanol solutions, embedded in Paraplast, and sectioned sagittally at 10 µm. The sections were stained with the Azan trichrome stain. To identify the species, reconstructions of the internal anatomy of the reproductive system were performed using a Zeiss Axio Scope A1 microscope. DNA extraction, amplification and sequencing DNA from four individuals (Table 1) was extracted using the phenol-chloroform protocol (Chen et al. 2010), using the tissue samples fixed in absolute ethanol. Once the DNA was extracted, its purity and concentration were calculated with a NanoDrop spectrophotometer (Thermo Fisher Scientific). Two partial sequences, one of approximately page 2 of 14Zoological Studies 62:15 (2023) © 2023 Academia Sinica, Taiwan Fig. 1. Bay of Archachon. Sampling sites. The green dots show the localities inside the bay, the red stars mark out the localities outside the bay. Arcachon Cap-Ferret Arguin Ile aux oiseaux 10 Km FRANCE SPAIN Atlantic Ocean Bay of Biscay 500 Km N Table 1. List of species included in the molecular analysis with their respective locality, GenBank accession number or museum catalogue number (MNCN) and reference Family Species 28S COI Locality Reference Discoceloidea Cryptocelidae Phaenocelis medvedica Marcus 1952 KY263701 -Brazil Bahia et al. 2017 Phaenocelis peleca Marcus and Marcus, 1968 MH700342 -Colombia Litvaitis et al. 2019 Phaenocelis purpurea (Schmarda, 1859) MH700347 -Curazao Litvaitis et al. 2019 Discocelidae Adenoplana evelinae Marcus, 1950 MH700268 -Panama Litvaitis et al. 2019 Discocelis tigrina (Blanchard, 1847) MN384690 MF993332 Spain Dittmann et al. 2019; Kenny et al. 2019 Discocelis sp.LC508146 LC508206 Japan Oya and Kajihara 2020 Ilyplanidae Ilyella gigas (Schmarda, 1859) LC508139 LC508199 Japan Oya and Kajihara 2020 Tripylocelis typica Haswell, 1907 MW377505 -Australia Rodríguez et al. 2021 Zygantroides serpulidicola Oya, Tsuyuki and Kajihara, 2020 -LC528161 Japan Oya et al. 2020 Leptoplanoidea Leptoplanidae Leptoplana tremellaris (Müller OF, 1773) KY263695 -Spain Bahia et al. 2017 Notocomplanidae Notocomplana hagiyai Oya and Kajihara, 2017 LC508129 LC176003 Japan Oya and Kajihara 2020; Oya and Kajihara 2017 Notocomplana humilis (Stimpson, 1857) LC100085 LC508204 Japan Tsunashima et al. 2017; Oya and Kajihara 2020 Notocomplana japonica (Kato, 1937) LC100087 LC176018 Japan Tsunashima et al. 2017; Oya and Kajihara 2017 Notocomplana koreana (Kato, 1937) LC100086 LC176014 Japan Tsunashima et al. 2017 Notocomplana septentrionalis (Kato, 1937) LC508130 LC176028 Japan Oya and Kajihara 2020; Tsunashima et al. 2017 Notoplanidae Notoplana australis (Schmarda, 1859) AY157153 -Australia Lockyer et al. 2003 Notoplana delicata Yeri and Kaburaki, 1918 LC100088 LC508205 Japan Tsunashima et al. 2017; Oya and Kajihara 2020 page 3 of 14Zoological Studies 62:15 (2023) © 2023 Academia Sinica, Taiwan Family Species 28S COI Locality Reference Pseudostylochidae Pseudostylochus elongatus (Kato, 1937) LC100083 LC508207 Japan Tsunashima et al. 2017; Oya and Kajihara 2020 Pseudostylochus intermedius Kato, 1939 LC508141 LC508201 Japan Oya and Kajihara 2020 Pseudostylochus obscurus (Stimpson, 1857) LC100084 LC508197 Japan Tsunashima et al. 2017; Oya and Kajihara 2020 Pseudostylochus takeshitai (Yeri and Kaburaki, 1918) LC508142 LC508202 Japan Oya and Kajihara 2020 Stylochoplanidae Amemiyaia pacifica Kato, 1944 LC508143 LC508203 Japan Oya and Kajihara 2020 Armatoplana leptalea (Marcus, 1947) KY263648 -Brazil Bahia et al. 2017 Comoplana pusilla (Bock, 1924) LC508134 LC508194 Japan Oya and Kajihara 2020 Stylochoplana sp. -KP259873 New Zealand Salvitti et al. 2015 Stylochoidea Hoploplanidae Hoploplana californica Hyman, 1953 KC869850 -USA Laumer and Giribet 2014 Hoploplana divae Marcus, 1950 KY263692 -Brazil Bahia et al. 2017 Hoploplana elisabelloi Noreña, Rodríguez, Pérez and Almón, 2015 -KT363735 -Aguado et al. 2016 Hoploplana ornata Yeri and Kaburaki, 1918 LC508135 LC508195 Japan Oya and Kajihara 2020 Hoploplana villosa (Lang, 1884) LC100076 -Japan Tsunashima et al. 2017 Idioplanidae Idioplana atlantica (Bock, 1913) MH700310 -Panama Litvaitis et al. 2019 Idioplana australiensis Woodworth, 1898 MW377499 MW375903 Australia Rodríguez et al. 2021 Idiostylochus tortuosus sp. nov. OM367916 OM365887 France This study Idiostylochus tortuosus sp. nov. OM367917 OM365888 France This study Idiostylochus tortuosus sp. nov. OM367918 OM365889 France This study Idiostylochus tortuosus sp. nov. OM367919 ON796529 France This study Idiostylochus tortuosus sp. nov. ON796524 ON796527 France This study Idiostylochus tortuosus sp. nov. ON796525 ON796528 France This study Idiostylochus tortuosus sp. nov. ON796526 ON796530 France This study Latocestidae Eulatocestus australis Rodríguez, Hutchings and Williamson, 2021 MW377502 MW375906 Australia Rodríguez et al. 2021 Latocestus plehni Laidlaw, 1906 MK299376 -Canary Islands Cuadrado et al. 2021 Latocestus plehni Laidlaw, 1906 MK299377 -Cabo Verde Cuadrado et al. 2021 Planoceridae Heteroplanocera katoi Oya and Kajihara, 2021 LC545568 LC545561 Japan Oya and Kajihara 2021 Paraplanocera oligoglena (Schmarda, 1859) LC545569 LC545563 Japan Oya and Kajihara 2021 Paraplanocera sp. KY263699 -Greece Bahia et al. 2017 Planocera multitentaculata Kato, 1944 LC100081 LC508192 Japan Tsunashima et al. 2017; Oya and Kajihara 2020 Planocera reticulata (Stimpson, 1855) LC508148 LC508208 Japan Oya and Kajihara 2020 Plehniidae Paraplehnia pacifica (Kato, 1939) LC508132 LC508193 Japan Oya and Kajihara 2020 Paraplehnia seisuiae Oya, Kimura and Kajihara, 2019 LC467000 LC466999 Japan Oya et al. 2019 Stylochidae Leptostylochus gracilis Kato, 1934 LC100078 -Japan Tsunashima et al. 2017 Leptostylochus cf. gracilis Kato, 1934 LC508138 LC508198 Japan Oya and Kajihara 2020 Leptostylochus victoriensis Beveridge, 2018 MW377495 MW375899 Australia Rodríguez et al. 2021 Mirostylochus akkeshiensis Kato, 1937 LC508149 LC508209 Japan Oya and Kajihara 2020 Stylochus cf. aomori Kato, 1937 LC508140 LC508200 Japan Oya and Kajihara 2020 Stylochus ijimai Yeri & Kaburaki, 1918 LC100079 -Japan Tsunashima et al. 2017 Stylochus neapolitanus (Delle Chiaje, 1841) MZ292841 - Spain Rodríguez et al. Unpublished Stylochus refertus Du Bois-Reymond Marcus, 1965 KY263694 -Brazil Bahia et al. 2017 Stylochus zebra (Verrill, 1882) AF342800 -USA Mallatt and Winchell 2002 Stylochus sp. KY263743 -Peru Bahia et al. 2017 Outgroup Cestoplanidae Cestoplana nopperabo Oya and Kajihara, 2018 LC322284 LC322283 Japan Oya and Kajihara 2018 Pericelidae Pericelis flavomarginata Tsuyuki, Oya, Jimi and Kajihara, 2020 LC568535 LC568538 Japan Tsuyuki et al. 2020 Table 1. (Continued) page 4 of 14Zoological Studies 62:15 (2023) © 2023 Academia Sinica, Taiwan 1,000 bp from the 28S gene and another of 700 bp from the COI gene, were amplified by PCR. For the 28S sequences, a forward primer (5'-AGCCCAGCACCGAATCCT-3') and a reverse primer (5'-GCAAACCAAGTAGGGTGTCGC-3') were used (Cuadrado et al. 2021). The reaction was carried out in a final volume of 25 µl with 1 μl of DNA, 12.5 μl of DreamTaq DNA polymerase and 1 μl of each primer. The amplification protocol used was an initial denaturation step at 95°C (4 min), followed by 35 cycles of denaturation at 95°C (1 min), annealing at 59°C (1 min) and extension at 72°C (1 min), with a final extension at 72°C (10 min). For the COI sequences, the primers Acotylea_ COI_F (5'-ACTTTATTCTACTAATCATAAGGATATA GG-3') and Acotylea_COI_R (5'-CTTTCCTCTATAAA ATGTTACTATTTGAGA-3') were used (Oya and Kajihara 2020). The reaction was carried out with the same volumes as those used for the 28S gene. The amplification protocol was as follows: initial denaturation step at 94°C (5 min), 35 cycles of denaturation at 94°C (30 s), annealing at 50°C (30 s) and extension at 72°C (1 min), with a final extension at 72°C (7 min). The PCR products were purified using ExoSAP (Bell 2008). The purified samples were sent to Secugen S.L. (www.secugen.es) for sequencing. Finally, the sequences obtained from the forward and reverse primers were combined and edited with Sequencher 4.1.4 (Gene Codes Corporation, Ann Arbor, MI, USA; http://www.genecodes.com). Sequence alignment and molecular analyses The different trees were obtained applying the Maximum Likelihood (ML) and Bayesian Inference (BI) methods, using the sequences obtained in this work and those available in GenBank (NCBI) of representative species of the main families of the suborder Acotylea (Table 1). Two Cotylea were used as outgroups: Pericelis flavomarginata Tsuyuki, Oya, Jimi and Kajihara, 2020 and Cestoplana nopperabo Oya and Kajihara, 2018 (Table 1). Sequence alignment was performed with MAFFT (Katoh et al. 2018) with the default options. Ambiguous regions were removed using Gblocks ver. 0.91b (Talavera and Castresana 2007) with the least restrictive options. The alignments were checked manually with BioEdit (Hall 1999). For this study, a dataset of 706 bp and 58 sequences for the 28S gene analysis and a dataset of 640 bp and 40 sequences for the COI gene analysis were used (the complete sequences of the new species can be found in GenBank, Table 1). The substitution model used for all the analyses was GTR+I+G, which was determined with ModelFinder (Kalyaanamoorthy et al. 2017) using the Akaike Information Criterion (AIC) (Akaike 1974). ML analyses were performed with IQ-TREE (Trifinopoulos et al. 2016). Nodal support was calculated with a bootstrap standard test with 1,000 replicates. BI analyses were performed with MrBayes 3.2.3 (Ronquist et al. 2012). Two simultaneous analyses of 10,000,000 generations were run with four chains (one cold, three heated) and a tree sampling frequency of 1,000. The convergence of the chains was determined using the value of the standard deviation of the frequencies (< 0.05). The first 25% of the trees were discarded as burn-in. The trees resulting from both methods were visualized and edited with iTOL ver. 6.3 (Letunic and Bork 2021). RESULTS SYSTEMATICS Order Polycladida Lang, 1881 Suborder Acotylea Lang, 1884 Superfamily Stylochoidea Poche, 1926 Family Idioplanidae Dittmann, Cuadrado, Aguado, Noreña and Egger, 2019 Idiostylochus gen. nov. urn:lsid:zoobank.org:act:B1874F30-7A5B-45C6-AD8A34A37FC66E5A Diagnosis: Idioplanidae with the pharynx in the middle of the body. Cerebral and marginal eyes present. Male copulatory apparatus with spermiducal bulbs and prostatic vesicle. Seminal vesicle absent. Female apparatus with a tubular Lang’s vesicle and cement glands. The vagina makes a posterior turn before reaching the penis papilla. Type species: Idiostylochus tortuosus sp. nov. Etymology: The name Idiostylochus derives from a combination of Idioplana and Stylochus, regarding the presence of a unique combination of characters found in part in these genera. Idiostylochus tortuosus sp. nov. (Fig. 2) urn:lsid:zoobank.org:act:BCF1166D-6D0A-4B79-899A59D3B262644C Type material: Holotype: 1 specimen. Arcachon Bay, France, October 20, 2020. Sagittal sections stained with Azan trichrome. MNCN 4.01/4263 to MNCN 4.01/4291 (29 slides). GeneBank accesion numbers: page 5 of 14Zoological Studies 62:15 (2023) © 2023 Academia Sinica, Taiwan ON796526 (28S), ON796530 (COI). Paratype: 1 specimen. Arcachon Bay, France, October 20, 2020. Sagittal sections stained with Azan trichrome. MNCN 4.01/4292 to MNCN 4.01/4335 (44 slides). Additional material: tissues preserved in 100% Ethanol and sagittal sections stained with Azan trichrome; Arcachon Bay, France, October 20, 2020. For GeneBank accession numbers see table 1. Diagnosis: Male copulatory apparatus with conspicuous spermiducal bulbs and a small prostatic vesicle. Elongated male atrium covered by glandular tissue. Female apparatus with a vagina bulbosa and well-developed cement glands that surround the two sections of the vagina (externa e interna). Etymology: The specific name derives from the Latin tortuous, due to the winding and complex course of the distal portion of the vasa deferentia and spermiducal bulbs. Description: Body shape rounded-oval with slightly undulated margins (Fig. 2A). Holotype 1.8 cm long and 1.1 cm wide, paratype 2.2 cm long and 1.5 cm wide. Body consistency firm and fleshy, more delicate and thinner towards the margins. Tentacles lacking. Cerebral and marginal eyes present. Background pigmentation chocolate brown to caramel in the margins. Numerous dark spots scattered over the dorsal surface, more abundant along the main body axis (Fig. 2A). Ventral surface pale, with grey to beige tonalities. Epithelium, basal membrane and body musculature more developed on the dorsal than on the ventral side (Fig. 2B). Pharynx ruffled, well developed, extends throughout the mid-body region. Oral pore at the beginning of the posterior body-half. Reproductive system: male and female reproductive organs are located directly after the pharynx, in the posterior half of the animal. Male copulatory apparatus consisting of a conical penis papilla (or peneal bulb) and a small pyriform prostatic vesicle, seminal vesicle absent (Fig. 2C, E). Vasa deferentia forms bulky spermiducal bulbs. The diameter of the bulbs decreases as they approach each other until they join forming the common vas deferens, which opens into the middle region of the ejaculatory duct. Both vasa deferentia and spermiducal bulbs follow a tortuous course. Free prostatic vesicle. Male atrium elongated and covered with glandular, spongy tissue. Female gonopore posterior to the male gonopore (Fig. 2D, E, F). With vagina bulbosa. The vagina externa curves anteriorly to the male reproductive system, then upwardly and continues posteriorly into the vagina interna. The oviduct opens between the vagina externa and interna. Connected to the vagina interna is an elongated and tubular Lang’s vesicle (Fig. 2D, E). The general appearance of the female apparatus is compact with small visible folds, mainly in the vagina externa, and surrounded by abundant cement glands. Biology and occurrence Idiostylochus tortuosus was found in oyster cultures of Magallana gigas. Individuals were collected living in the mantle cavity of diseased or dead oysters as well as swimming around the oyster farming devices. Some individuals have also been observed feeding on natural beds of Mytilus edulis Linnaeus, 1758 and Mytilus galloprovincialis Lamarck, 1819 near the oyster farms (Vieira and Nowaczyk pers. com.). Although the presence of Idiostylochus was known long ago by oyster farmers, in recent years, the frequency and number of polyclads specimens seem to have increased, and their presence has caused noticeable damage to oyster and mussel crops. Taxonomical remarks The superfamily Stylochoidea, where the new species Idiostylochus tortuosus was placed, presents a free prostatic vesicle (Faubel 1983). From the molecular point of view, the closest related genera (see - tree 28S, Fig. 4) are Idioplana Woodworth, 1898 (Idioplanidae), Leptostylochus Bock, 1925 (Stylochidae), and also, but less related, Mirostylochus Kato, 1937 (Stylochidae), Latocestus Plehn, 1896 and Eulatocestus Faubel, 1983 (Latocestidae). All these genera present a free prostatic vesicle and either developed spermiducal bulbs or an elongated seminal vesicle. The new species shares some characters with these genera, while others are clearly different. A comparative discussion follows. Leptostylochus (Stylochidae) (Fig. 3A) is characterized by an elongated slender body shape; tentacular, cerebral, marginal and often frontal eyes; male copulatory apparatus with an unarmed penis papilla and without seminal vesicle, but with spermiducal bulbs that join into a common vas deferens before entering the medial region of the ejaculatory duct; female reproductive system with a developed Lang’s vesicle (Kato 1934; Faubel 1983; Beveridge 2017). Idiostylochus gen. nov. resembles Leptostylochus because of the presence of large spermiducal bulbs. Furthermore, the female system shows common features, like the well-developed shell glands around the vagina externa. In contrast, the Lang’s vesicle is conspicuous in Leptostylochus and reduced in Idiostylochus, which appears as a small tubular duct. Latocestus and Eulatocestus (Latocestidae) share a similar morphology. Both genera have spermiducal bulbs, unarmed penis papilla and a page 6 of 14Zoological Studies 62:15 (2023) © 2023 Academia Sinica, Taiwan vp CD E op mp fp ph mp fp spb ovd Lv shg ve vi pv ph vd F Fig. 2. Idiostylochus tortuosus gen. nov., sp. nov. A–B, Live specimen. A, dorsal view; B, ventral view. C–D, histological section through male (left) and female (right) copulatory organs. E, sagittal reconstruction of the reproductive system. F, sagittal reconstruction of whole animal (anterior end at the left). Scale bars = 500 µm. page 7 of 14Zoological Studies 62:15 (2023) © 2023 Academia Sinica, Taiwan simple female apparatus with Lang’s vesicle (Plehn 1896; Faubel 1983) (Fig. 3B). The main difference between Latocestus and Eulatocestus is the lining of the prostatic vesicle: irregular or fingered in Latocestus and a web of glandular follicles in Eulatocestus. Both genera are distinguishable from Idiostylochus by the morphology of the female apparatus. The presence of a well-developed Lang’s vesicle and a simple, nonbulbous vagina with much less abundant cement glands differentiates both genera from Idiostylochus. Idioplana (Idioplanidae) is characterized by a male copulatory organ with an unarmed penis papilla, prostatic and seminal vesicle; female apparatus extended over the male apparatus; and an anchor-shaped Lang’s vesicle (Woodworth 1898; Meixner 1907; Faubel 1983; Rodríguez et al. 2021) (Fig. 3C). The analysis of the morphological data reveals external anatomical similarities, such as the oval body shape, the reddish-brown dorsal and whitish ventral coloration and the arrangement of the cerebral eyes, but both genera are clearly differentiated by the presence of conspicuous tentacles in Idioplana, absent in Idiostylochus, and the differences between the reproductive systems. In Idiostylochus the seminal vesicle is absent, the female canal is shorter, since the vagina interna do not extend anteriorly over the male copulatory organ, and the Lang’s vesicle is tubular. Within the male apparatus, there are clear differences. In Idioplana a seminal vesicle is present, while in Idiostylochus is absent. In Idiostylochus the function of the seminal vesicle is carried out by the spermiducal bulbs, since the latter replace the former in its absence. Although the molecular analysis clusters Mirostylochus (Stylochidae) near Idiostylochus, the two genera are morphologically distinct. Mirostylochus is characterized by tentacular, cerebral and marginal eyes; and a female apparatus with ductus vaginalis, from which the vagina interna opens to the exterior behind the female gonopore. Lang’s vesicle is absent (Kato 1937; Tokinova 2003). Idiostylochus gen. nov. and Mirostylochus differ in the morphology of the female apparatus, as Idiostylochus possesses a tubular Lang’s vesicle and lacks ductus vaginalis. Molecular approach The molecular studies are based on two datasets of the genes 28S (nuclear) and COI (mitochondrial). The methods applied for the analyses were Maximum Likelihood (ML) and Bayesian Inference (BI). Within the tree generated during the analysis of 28S (Fig. 4) we can distinguish three wellsupported branches, the clusters of the superfamilies Discoceloidea, Leptoplanoidea and Stylochoidea Fig. 3. Comparison of the copulatory apparatuses of different genera of Stylochoidea. A, Leptostylochus Bock, 1925; B, Latocestus Plehn, 1896; C, Idioplana Woodworth 1898. B C A Idioplana Leptostylochus Latocestus Lv Lv Lv pv pv pv shg shg shg sv spb spb spb ve ve ve vi vi vi page 8 of 14 Zoological Studies 62:15 (2023) © 2023 Academia Sinica, Taiwan Fig. 4. Bayesian / Maximum likelihood tree based on partial sequences of the 28S gene (total length: 706 bp). Numbers in the nodes correspond to the posterior probability values of the BI analysis (> 0.70) and bootstrap support values of the ML analysis (> 50), respectively. Cestoplana nopperabo Illyella gigas Discocelis tigrina Adenoplana evelinae Discocelis sp. Phaenocelis peleca Amemiyaia pacifica Phaenocelis purpura Phaenocelis medvedica Armatoplana leptalea Leptoplana tremellaris Notoplana australis Notoplana delicata Notocomplana humilis Notocomplana koreana Notocomplana japonica Notocomplana hagiyai Notocomplana septentrionalis Tripylocelis typica Pseudostylochus intermedius Pseudostylochus obscurus Pseudostylochus elongatus Pseudostylochus takeshitai Comoplana pusilla Stylochus cf aomori Stylochus neapolitanus Stylochus sp. Stylochus zebra Stylochus ijimai Stylochus refertus Paraplehnia seisuiae Paraplehnia pacifica Hoploplana villosa Hoploplana californica Hoploplana ornata Hoploplana divae Heteroplanocera katoi Paraplanocera oligoglena Paraplanocera sp. Planocera reticulata Planocera multitentaculata Idioplana atlantica Idioplana australiensis Idiostylochus tortuosus Leptostylochus cf. gracilis Leptostylochus cf. gracilis Leptostylochus victoriensis Mirostylochus akkeshiensis Latocestus plehni Eulatocestus australis Latocestus plehni 100/96 100/91 100/86 0.93/75 0.98/- 1.00/100 1.00/100 1.00/100 1.00/100 1.00/100 1.00/100 1.00/ 100 1.00/83 0.96/- 0.93/- 1.00/88 0.98/- 1.00/100 0.98/68 0.99/68 0.81/- 1.00/99 Tree scale: 0.1 1.00/88 1.00/96 0.93/57 1.00/ 1.00/78 1.00/91 1.00/88 1.00/99 1.00/ 90 0.98/92 1.00/94 0.84/- 0.88/- 0.81/- DISCOCELOIDEA LEPTOPLANOIDEA STYLOCHOIDEA Stylochidae Plehniidae Hoploplanidae Planoceridae Idioplanidae Latocestidae page 9 of 14Zoological Studies 62:15 (2023)