Zoosyst. Evol. 100 (3) 2024, 877-895 | DOI 10.3897/zse.100.120244 ee Ee BERLIN Marine microturbellarians from Japan, with descriptions of two new species of Reinhardorhynchus (Platyhelminthes, Rhabdocoela, Koinocystididae) Aoi Tsuyuki!*, Jnoe Reyes?, Yuki Oya*, Kevin C. Wakeman?®, Brian S. Leander’, Niels W. L. Van Steenkiste’® Faculty of Science, Hokkaido University, Sapporo, Hokkaido, 060-0810, Japan Creative Research Institution, Hokkaido University, Sapporo, Hokkaido, 001-0021, Japan Facultad de Ciencias de la Vida y de la Salud, Universidad Cientifica del Sur, Lima, Peru College of Arts and Sciences, J. F. Oberlin University, 3758 Tokiwa, Machida, Tokyo, 194-0294, Japan Institute for the Advancement of High Education, Hokkaido University, Sapporo, Hokkaido, Japan Graduate School of Science, Hokkaido University, Sapporo, Hokkaido, 080-0810, Japan Departments of Botany and Zoology, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada Hakai Institute, Heriot Bay, Quadra Island, BC, VOP 1H0, Canada CON DO FP WY https://zoobank. org/C025A8A6-116F-4BAF-94AA-6276BC84D2C8 Corresponding author: Jhoe Reyes (jreyesp@cientifica.edu.pe) Academic editor: Pavel Stoev # Received 5 February 2024 Accepted | April 2024 Published 3 July 2024 Abstract Marine microturbellarians are an assemblage of meiofaunal flatworms abundant in sediments and on seaweeds around the world. The diversity and distribution of these animals in Japan are poorly understood. Here, we provide an overview of all recorded spe- cies in Japan and characterize two new species of the rhabdocoel genus Reinhardorhynchus based on morphological features and a molecular phylogeny inferred from 18S and 28S rDNA sequences. Reinhardorhynchus ryukyuensis sp. nov. can be distinguished from other species in the genus by the lack of an armed cirrus and by the presence of two larger opposing hooks and five smaller interconnected hooks in its male copulatory organ. Reinhardorhynchus sagamianus sp. nov. differs from its congeners because its male copulatory organ combines a bipartite cirrus armed with a belt of overlapping scale-like spines, an unarmed accessory cirrus, and two large distal accessory hooks. Our molecular phylogenetic analyses show that R. rvukyuensis sp. nov. and R. sagamianus sp. nov. form a clade with all the other species of Reinhardorhynchus for which DNA sequence data are available. Within this clade, R. sagamianus sp. nov. is in a clade that also includes R. riegeri and R. anamariae. The discovery of these new species highlights the importance of uncovering and documenting the hidden biodiversity along Japan’s coastal margin. Key Words Distribution, flatworms, Japanese invertebrates, Kalyptorhynchia, marine meiofauna Introduction Microturbellarians are microscopic and mostly free-living flatworms that are common in marine meiofaunal com- munities around the globe (Schockaert et al. 2008; Armo- nies 2017; Fegley et al. 2020). They inhabit various types of interstitial substrates (e.g., algae and sediments) in in- tertidal and subtidal habitats and have also been recorded at depths of up to ~600 m (Artois et al. 2000; Aramayo 2018; Armonies 2023). Our understanding of the diversi- ty and distribution of marine microturbellarians is most- ly limited to regions where dedicated research has been conducted on these animals, including the coastal areas in Europe (e.g., Casu et al. 2014; Schockaert 2014; Gobert Copyright Tsuyuki, A. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 878 Tsuyuki, A. et al.: Two new species of Koinocystididae, Rhabdocoela, from Japan et al. 2020; Armonies 2023), Brazil (e.g., Marcus 1950, 1951, 1952; Braccini et al. 2016), Cuba (e.g., Diez et al. 2018, 2023a, 2023b), or Canada (e.g., Van Steenkiste and Leander 2018a, 2018b; Stephenson et al. 2019). Howev- er, even in some of these well-studied areas, the diversity of marine microturbellarians can be significantly higher than initial studies have shown. For example, ecological estimations suggest the presence of ~200 species in Cuba (Diez et al. 2023b) and ~400 species on the island of Sylt (northern Germany, in the North Sea) (Armonies 2023). For Japan, only scattered records of marine and brack- ish water microturbellarians are known from the litera- ture. The first marine microturbellarians described from Japan were prolecithophorans (Tozawa 1918). Since the 1950’s, new representatives of Macrostomorpha, Rhab- docoela, Proseriata, and Prolecithophora have been re- ported intermittently (Westblad 1955; Karling 1966; Taji- ka 1977, 1978, 1979, 1980, 1981a, 1981b, 1982a, 1982b, 1982c, 1983a, 1983b, 1983c, 1984; Ax 2008; Omi 2018, 2020; Takeda and Kayjihara 2018). However, these studies are confined to a limited number of localities in Japan. With numerous islands and inlets bordering the Sea of Okhotsk, the Sea of Japan, the East China Sea, the Phil- ippines Sea, and the Northwest Pacific Ocean, the coastal margin of Japan represents an important but poorly ex- plored part of the wider Pacific Ocean. It is expected that 50° H 40° 30° §) Macrostomorpha | Rhabdocoela !)) Prolecithophora se || Proseriata a 120° 130° 140° the diversity of marine microturbellarians in Japan is far from being adequately described. Here, we characterize two new species of Koinocyst- ididae (Rhabdocoela) with morphological and molecular data. Their phylogenetic positions are determined based on analyses using 18S and 28S rDNA sequences. Addi- tionally, we provide a concise overview of the marine and brackish microturbellarian diversity of Japan and high- light the importance of such research. Materials and methods Specimen collection and fixation The specimens of Reinhardorhynchus ryukyuensis sp. nov. were collected by Niels Van Steenkiste and Kevy- in Wakeman at Onna, Okinawa, Japan (26°28'52.7"N, 127°50'18.8"E) in February 2019 from a coarse mix- ture of sand, coral fragments, and shell hash in seagrass meadows in a shallow intertidal bay. The specimens of Reinhardorhynchus sagamianus sp. nov. were collected by Aoi Tsuyuki and Yuki Oya at Sangashita beach, Haya- ma, Kanagawa (35°15'58.3"N, 139°34'19.64"E) in April and August 2023, from clean, coarse sandy sediments in the upper intertidal zone (Fig 1). The upper centimeters 150° 140° Figure 1. Records of marine microturbellarians in Japan. A. Map shows the documented occurrences of marine microturbellarians reported from Japan. The size and numbers within the pie chart represent the number of recorded species from a) the Okhotsk Sea coast of Hokkaido, b) the Sea of Japan coast of Hokkaido, c) the Pacific coast of Hokkaido, d) Mutsu Bay and the brackish water ar- eas of Aomori, and e) the Pacific coast of Kanagawa. The small circles without numbers indicate single species from the Inland Sea, East China, and the Pacific coasts of Okinawa and Ishigaki Islands, respectively. B. Magnification of the area, including Okinawa, with a star designating the type locality of Reinhardorhynchus ryukyuensis n. sp. C. Magnification of the area, including Kanagawa, with a star designating the type locality of R. sagamianus sp. nov. zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 877-895 of sediment were collected using a shovel. Specimens of R. ryukyuensis sp. nov. were separated from sediments using the MgCl, decantation method (Schockaert 1996). Individual worms were isolated under a stereoscope and whole-mounted alive in seawater to be studied and pho- tographed at the Okinawa Institute of Science and Tech- nology (OIST) under a compound microscope (Zeiss Axioscope) equipped with DIC. Two specimens were whole-mounted in lactophenol to study the sclerotized parts of the male copulatory organ. Two additional spec- imens were frozen in 3 ul of filtered seawater for DNA extraction. The specimens of R. sagamianus sp. nov. were procured by meticulously rinsing the collected san- dy substrate with seawater, employing a dip net with an approximately 1-mm mesh size. Subsequently, the speci- mens were pipetted into petri dishes to facilitate isolation, whole-mounted in seawater to observe morphological characters on two life specimens, mounted in Entellan New (Merck) to study the sclerotized structures, or fixed in 99.5% ethanol for DNA extraction. Morphological observations Measurements and descriptions were made based on squashed preparations. Measurements of the sclerotized structures such as hooks and spines, as well as for soft body tissues, are expressed in micrometers (um) and were taken using ImageJ software (Schneider et al. 2012). The sclerotized structures in live and fixed specimens were photographed with a Nikon D5600 digital camera affixed to an Olympus BX51 light microscope and with an Olym- pus DP20 digital camera affixed to an Olympus BX21 compound microscope. Figures were created with Adobe Illustrator CC 23.0.3 (Adobe Systems Inc., USA). All the whole mounted specimens fixed in lactophenol and Entel- lan New were deposited at the National Museum of Na- ture and Science, Tokyo (NSMT). A comprehensive bib- liographic compilation of historic records and distribution data of free-living microturbellarians from marine and brackish water environments in Japan was also conducted. DNA extraction, polymerase chain reaction, and sequencing Total genomic DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s in- structions. For phylogenetic inference, fragments of the 18S rDNA and 28S rDNA were PCR amplified using the primers and thermocycling conditions in Table 1. For the 18S and 28S rDNA of R. sagamianus sp. nov., 10- ul reaction volumes were used, each of which contained 1 ul of total DNA template, 1 ul of 10 x ExTaq buffer (Takara Bio), 2 mM of each dNTP, 1 uM of each prim- er, and 0.25 U of Takara Ex Taq DNA polymerase (5 U/ ul; Takara Bio) in deionized water. For the 18S and 28S rDNA of R. ryukyuensis sp. nov., Illustra™ PuReTaq™ 879 Ready-To-Go™ PCR beads (GE Healthcare) were sus- pended in a 25-ul volume of water, primers (0.2 uM), and DNA template (1.5 ul). Amplicons were visualized on 1.5% agarose gels stained with GelRed™ (Biotium) (R. ryukyuensis sp. nov.) or 1.0% agarose gels stained with FluoroDye DNA Fluorescent Loading Dye (SMO- BIO) (R. sagamianus sp. nov.) and purified enzymati- cally using Illustra™ ExoProStar S (GE Healthcare) (R. ryukyuensis sp. nov.) or Exonuclease I and SAP (Takara Bio) (R. sagamianus sp. nov.). Amplicons of R. ryukyuen- sis Sp. nov. were subsequently sequenced by Genewiz (Azenta Life Sciences) through standard Sanger DNA sequencing, while amplicons of R. sagamianus sp. nov. were sequenced with a BigDye Terminator Kit ver. 3.1 and a 3730 Genetic Analyzer (Life Technologies), using the amplification and internal sequencing primers shown in Table 1. Trace files were assembled into full sequenc- es in either Geneious v11.0.15 (Kearse et al. 2012) or MEGA ver. 7.0 (Kumar et al. 2016) and subjected to a BLAST search on the NCBI website (http://blast.ncbi. nlm.nih.gov) to verify the specimens’ taxonomic identi- ty. Sequences were deposited in DDBJ/EMBL/GenBank, with accession numbers provided in Table 2. Molecular phylogenetic analyses For phylogenetic analyses, a concatenated dataset (3,264 bp) comprising partial 18S rDNA (1,642 bp) and 28S rDNA (1,622 bp) was prepared using DNA sequences of 24 koinocystidids in addition to the sequences of two individuals of Reinhardorhynchus ryukyuensis sp. nov. and one individual of R. sagamianus sp. nov. (Table 2). Cysti- plex axi Karling, 1964, and Cystiplex sp. were included as outgroup taxa. Sequences were aligned using MAFFT ver. 7.472 (Katoh et al. 2019) with the L-INS-I strategy selected under the “Auto” option. Ambiguous sites were trimmed with Clipkit ver. 1.0 using the “kpic” option (Steenwyk et al. 2020). The optimal substitution models selected with PartitionFinder ver. 2.1.1. (Lanfear et al. 2016) were GTR+I+G for both the 18S and 28S rDNA partitions. A maximum likelihood (ML) analysis was performed using IQTree ver. 1.6 (Nguyen et al. 2015) under a partition mod- el (Chernomor et al. 2016). Bayesian inference (BI) of the phylogeny was performed using MrBayes ver. 3.2.3 (Ron- quist and Huelsenbeck 2003; Altekar et al. 2004) with two independent runs of Metropolis-coupled Markov chain Monte Carlo (MCMC), each consisting of four chains of 1,000,000 generations. All parameters (statefreq, revmat, shape, and pinvar) were unlinked between each position; trees were sampled every 100 generations. The first 25% of the trees were discarded as burn-in before a 50% ma- jority-rule consensus tree was constructed based on the re- maining 7,500 trees. Convergence was confirmed with the average standard deviation of split frequencies (0.008833), potential scale reduction factors for all parameters (0.999— 1.006), and effective sample sizes for all parameters (=322). Nodal support within the ML tree was assessed by analyses zse.pensoft.net 880 Tsuyuki, A. et al.: Two new species of Koinocystididae, Rhabdocoela, from Japan Table 1. Primers and thermocycling conditions used in this study. Primers R. ryukyuensis sp. nov. Primer name Sequence (5‘-3") Application Reference 18S rDNA TimA AMCTGGTTGATCCTGCCAG Amplification and Norén and Jondelius (1999) sequencing 18S rDNA TimB TGATCCATCTGCAGGTTCACCT Amplification and Norén and Jondelius (1999) sequencing 18S rDNA 600F GGTGCCAGCAGCCGCGGT Sequencing Norén and Jondelius (1999) 18S rDNA 600R ACCGCGGCTGCTGGCACC Sequencing Norén and Jondelius (1999) 18S rDNA 1100F CAGAGGTTCGAAGACGATC Sequencing Norén and Jondelius (1999) 18S rDNA 1100R GATCGTCTTCGAACCTCTG Sequencing Norén and Jondelius (1999) 18S rDNA 18S7F GCAATAACAGGTCTGTGATGC Sequencing Norén and Jondelius (1999) 18S rDNA 18S7FK GCATCACAGACCTGTTATTGC Sequencing Norén and Jondelius (1999) 28S rDNA LSU5 TAGGTCGACCCGCTGAAYTTA Amplification and Littlewood et al. (2000) sequencing 28S rDNA LSUD6-3B GCTGTTCACATGGAACCCTTCTC Amplification and Van Steenkiste et al. (2013) sequencing 28S rDNA L300F CAAGTACCGTGAGGGAAAGTTG Sequencing Littlewood et al. (2000) 28S rDNA L300R CAACTTTCCCTCACGGTACTTG Sequencing Littlewood et al. (2000) 28S rDNA L1200F CCCGAAAGATGGTGAACTATG Sequencing Littlewood et al. (2000) 28S rDNA L1200R GCATAGTTCACCATCTTTCGG Sequencing Littlewood et al. (2000) R. sagamianus sp. nov. 18S rDNA hrms18S_F ATCCTGCCAGTAGTCATATGC Amplification and Oya and Kajihara (2020) sequencing 18S rDNA hrms18S_Fil GCCGCGGTAATTCCAG Sequencing Oya and Kajihara (2020) 18S rDNA hrms18S_R CTACGGAAACCTTGTTACGAC Sequencing Oya and Kajihara (2020) 18S rDNA hrms18S_Ril © CTTTAATATACGCTATTGGAGCTGG Sequencing Oya and Kajihara (2020) 18S rDNA hrms18S_Ri2 CTATTTAGTGGCTAGAGTCTCGTTCG Amplification and Oya and Kajihara (2020) sequencing 28S rDNA LSU5 TAGGTCGACCCGCTGAAYTTA Amplification and Littlewood et al. (2000) sequencing 28S rDNA Rd4.8a ACCTATTCTCAAACTTTAAATGG Sequencing Whiting (2002) 28S rDNA rD5b CCACAGCGCCAGTTCTGCTTAC Sequencing Whiting (2002) 28S rDNA LSUD6-3B GCTGTTCACATGGAACCCTTCTC Amplification and Van Steenkiste et al. (2013) sequencing Thermocycling conditions R. ryukyuensis 18S rDNA 95 °C for 3m, touch down in 9 cycles (94 °C for 30 s, 60 °C down to 56 °C for 30 s, 72 °C for 1 m 30 s), 31 cycles (94 °C for 30s, 55 °C for 30s, 72 °C for 1 m 30s), 72 °C for 5m 28S rDNA 95 °C for 3m, touch down in 9 cycles (94 °C for 30 s, 60 °C down to 56 °C for 30 s, 72 °C for 1 m 30 s), 31 cycles (94 °C for 30 s, 55 °C for 30s, 72 °C for 1 m 30s), 72 °C for 5m R. sagamianus 18S rDNA 28S rDNA of 1,000 pseudoreplicates of ultrafast bootstrap (UFBoot) (Minh et al. 2013) and SH-aLRT branch tests (Guindon et al. 2010). ML UFBoot values >95%, SH-aLRT values >85%, and posterior probability (PP) values >0.90 were considered to indicate clade support. Abbreviations used in Figures a: apicomplexan; br: brain; bs: bursal stalk; bu: bursa; cg: common gonopore; cds: spines of the distal part of zse.pensoft.net 94 °C for 1 m, 35 cycles (94 °C for 30 s, 50 °C for 30s, 72 °C for 2 m), 72 °C for 7m 94 °C for 1m, 35 cycles (94 °C for 30s, 50 °C for 30s, 72 °C for 1.5 m), 72 °C for 7m the spiny belt; ciu: unarmed accessory cirrus; cia: armed cirrus; cm: circular muscles; cms: spines of the middle part of the spiny belt; cps: spines of the proximal part of the spiny belt; ed: ejaculatory duct; fa: female atrium; fd: female duct; fg: female glands; h: hook; i: intestine; ilm: inner layer of longitudinal muscles; lh: larger hooks; ma: male genital atrium; oe: oesophagus; olm: outer layer of longitudinal muscles; om: oblique muscles; ov: ovary; pg: prostate glands; ph: pharynx; pp: penis papilla; pr: proboscis; s: spine; sh: smaller hook; scl: sclerotized lay- er; Sv: seminal vesicle; t: testis; u: uterus; vi: vitellaria. Zoosyst. Evol. 100 (3) 2024, 877-895 Table 2. List of species and respective GenBank accession numbers used for the molecular phylogenetic analyses in this study. Species [taipusa divae Itaipusa biglandula [taipusa karlingi taipusa novacaledonica [taipusa sp. 1 Koinogladius sinensis YTP1 Koinogladius sinensis YTP2 Koinogladius sinensis YTP3 Mesorhynchus terminostylis Reinhardorhynchus anamariae Reinhardorhynchus hexacornutus Reinhardorhynchus riegeri Reinhardorhynchus riegeri (CU1272) Reinhardorhynchus ryukyuensis sp. nov. Reinhardorhynchus ryukyuensis sp. nov. Reinhardorhynchus sagamianus sp. nov. Reinhardorhynchus tahitiensis A Reinhardorhynchus tahitiensis B Rhinolasius dillonicus Sekerana Stolci Utelga heinckel Utelga heinckei (QU4) Utelga heinckei (QU43) Utelga heinckei (QU44) Utelga pseudoheinckel Koinocystididae sp. 1 Outgroup Cystiplex axi Cystiplex sp. Results Taxonomic Account Rhabdocoela Ehrenberg, 1831 Kalyptorhynchia von Graff, 1905 Eukalyptorhynchia Meixner, 1928 Koinocystididae Meixner, 1924 Reinhardorhynchus Diez, Monnens, & Artois, 2021 Reinhardorhynchus ryukyuensis Van Steenkiste, Wakeman, & Leander, sp. nov. https://zoobank.org/57D2EE7F-0934-4BB1-AF9A-84E1D0D804FB Fig. 2 Material examined. Holotype: JAPAN °1; Okinawa Pre- fecture, Onna; 26°28'52.7"N, 127°50'18.8"E; Feb. 2019; coarse mixture of sand, coral fragments, and shell hash from an intertidal seagrass bed; Niels Van Steenkiste and Kevin Wakeman leg.; one individual worm in a single slide [Holotype: NSMT-PI 6458]: Paratype: JAPAN °1; locality same as for holotype; Feb. 2019; Niels Van Steenkiste and Kevin Wakeman leg.; one individual worm in a single slide; [Paratype: NSMT-PI 6459]. 18S rDNA 28S rDNA MW081596 MW054455 MW081601 MW054460 MW081598 MW054457 KJ887481 KJ887528 KJ887451 KJ887557 MF443159 MF443174 MF443160 MF443175 MF443161 MF443176 AY775741 KJ887500 MW081597 MW054456 MW054464 MW054451 MW081595 MW054454 OR490859 OR490875 LC807766 LC807768 - LC807769 LC807767 LC807770 MW054463 MW054452 MW054462 MW054453 MW081602 MW054461 7 KJ887537 MW081600 MW054459 OR490861 OR490876 OR490862 - OR490863 OR490877 MW081599 MW054458 KR339027 - KJ887437 KJ887549 KJ887469 KJ887495 Other material. JAPAN °1; locality same as for holo- type; Feb. 2019; Niels Van Steenkiste and Kevin Wake- man leg.; two genomic DNA extracts from two individ- uals stored at -20 °C; GenBank: LC807766 (18S rDNA; 1,760 bp), LC807768, LC807769 (28S rDNA; 1,675 bp). Type locality. Japan, Okinawa Prefecture, Onna (26°28'52.7"N, 127°50'18.8"E). Diagnosis. Species of Reinhardorhynchus with con- juncta-duplex type male copulatory organ composed of a proximal globular part, a weakly sclerotized cylindri- cal middle part, and a distal penis papilla. Sclerotized structures of the copulatory organ consist of two large, separate hooks at the transition between the middle part and penis papilla and a distal girdle of two semi-elliptical plates bearing five smaller hooks. One larger hook with collared striated base, 29-31 um long, pointing proximal- ly; the other larger hook straight, with striated base, 29— 34 um long, pointing distally. The smaller distal hooks are 9-17 um long. Female system with bipartite female duct, muscular bursal stalk, large bursa, and pouch of fe- male glands. Description. General morphology. Animals are 840— 1060 um long (x = 935 um; n = 4), transparent, and have two eyes (Fig. 2A, B). General organization and internal morphology are consistent with other species of Reinhar- dorhynchus, as described by Diez et al. (2021). The large, zse.pensoft.net 882 Tsuyuki, A. et al.: Two new species of Koinocystididae, Rhabdocoela, from Japan Figure 2. Reinhardorhynchus ryukyuensis sp. nov. A, B. Micrograph and drawing of a live animal. C. Detail of the atrial organs in a live specimen. D. Male copulatory organ in a whole-mounted specimen fixed in lactophenol. E, F. Drawing and micrograph of the sclerotized parts of the male copulatory organ in the holo/paratype. G, H. Drawing and micrograph of the sclerotized parts of the male copulatory organ in the holo/paratype. zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 877-895 typical koinocystidid proboscis (pr, Fig. 2A, B) is about 1/4 of the body length (215—233 um; x = 224 um; n= 3), with a well-developed juncture sphincter. Ciliated cellu- lar epidermis with needle-like rhabdites is present all over the body. The globular pharynx (ph, Fig. 2A, B) is at about 50% of the body length, followed by an intestine (1, Fig. 2A, B) that runs all the way to the posterior end. The oesophagus (oe, Fig. 2A, B) is visible as a clear, transparent zone bor- dered by oesophageal glands right behind the pharynx. Male reproductive system. Paired testes are located in front of the pharynx (t, Fig. 2A, B). The conjuncta-duplex type male copulatory organ (165-195 um; xX = 180 um; n = 4) is inverted-pear shaped and encompasses the prostate vesicle (pg, Fig. 2A—C). This prostate vesicle is composed of gland necks of prostate glands originating extracap- sularly, extending into the middle part of the copulatory organ, and opening distally into the ejaculatory duct. The bulbous proximal part of the prostate vesicle is surrounded by circular muscles (cm, Fig. 2D), while the cylindrical middle part is surrounded by two layers of oblique mus- cles (om, Fig. 2D) and an inner layer of longitudinal mus- cles (ilm, Fig. 2D). The walls of the middle and distal parts of the copulatory organ are weakly sclerotized (scl, Fig. 2C, D) and distally form a penis papilla (pp, Fig. 2C, D), which enters into the male genital atrium (ma, Fig. 2C, D). The entire copulatory organ is encased in an outer layer of longitudinal muscles (olm, Fig. 2C, D). Paired semi- nal vesicles (sv, Fig. 2A—C) merge right before entering the copulatory organ proximally and form the ejaculatory duct (ed, Fig. 2D). The necks of the prostate glands and the ejaculatory duct run throughout the entire length of the copulatory organ and into the penis papilla. The male copulatory organ is provided with sclerotized structures consisting of two larger separate hooks and a gir- dle of five smaller interconnected hooks (Ih1—-lh2, sh1—sh5, Fig. 2E-H). The two larger hooks are positioned at the tran- sition of the cylindrical middle part to the penis papilla and point in opposite directions. The proximally pointing larger hook (lhl, Fig. 2E—H) measures 29-31 um (n = 2) and is provided with a collared striated base. The distally pointing larger hook (1h2, Fig. 2E—H) is straight, measures 29-34 um (n = 2), and has a striated base that seems to be continuous with the weakly sclerotized layer. The five distal, smaller hooks are connected by a complex sclerotized girdle sur- rounding the distal tip of the penis papilla. The smaller hooks shl (15-17 wm; n = 2) and sh2 (10-13 um; n = 2), and sh3 (10-13 um; n = 2) and sh4 (9-12 um; n = 2) are each connected through a semi-elliptical base, respectively. The base of the smaller hook sh5 (10-12 um; n = 2) seems to connect both semi-elliptical bases into an open girdle. Female reproductive system. The female structures are located caudal to the pharynx and include paired ova- ries (ov, Fig. 2B, C), a female duct (fd, Fig. 2B) consist- ing of two parts (fd1 and fd2, Fig. 2C), and a very muscu- lar bursal stalk (bs, Fig. 2B—D) guarding the entrance to a large bursa (bu, Fig. 2B, C). At its proximal end, the fe- male atrium (fa, Fig. 2B, C) receives the female duct, the 883 bursal stalk, and a pouch of female glands (fg, Fig. 2B, C). The female duct is separated from the female atrium by a small sphincter. A uterus (u, Fig. 2B) is present be- tween the male and female atria. Vitellaria and the com- mon gonopore could not be observed in the live animals. Etymology. Species epithet based on its occurrence on the Ryukyu Islands. Distribution. Okinawa Islands, Japan. Reinhardorhynchus sagamianus Tsuyuki, Reyes, Oya, & Van Steenkiste, sp. nov. https://zoobank. org/B2534A FD-346 1 -4329-A62F-3C6AC8467FB2 Fig. 3 Material examined. Holotype: JAPAN °1; Kanagawa Prefecture, Hayama, Sangashita beach; (35°15'58.3"N, 139°34'19.6"E); 21 April 2023; sandy substrates; Aoi Tsuyuki and Yuki Oya leg.; one individual worm in a sin- gle slide; [Holotype: NSMT-PI 6460]. Paratype: JAPAN °1; locality same as for holotype; 30 Aug 2023; Yuki Oya leg.; genomic DNA extract from one individual stored at -20 °C; GenBank: LC807767 (18S rDNA; 1,654 bp), LC807770 (28S rDNA; 1,667 bp); [Paratype: NSMT-DNA 56985]. Type locality. Japan, Kanagawa Prefecture, Hayama, Sangashita Beach (35°15'58.3"N, 139°34'19.6"E). Diagnosis. Species of Reinhardorhynchus with a cop- ulatory organ encompassing an armed cirrus, an unarmed accessory cirrus, and two distal hooks. Bipartite armed cirrus consisting of two sacs lined with a continuous +295.2-um-long sclerotized belt of overlapping scale- like spines. Larger sac with more spaced-out, triangular, +20.1 um-long spines on the proximal end of the belt. Spines gradually decrease in size distally as the belt runs towards and folds into the smaller sac, increasing in size (+6.1 to +22.2 um long) towards the proximal tip of the smaller sac, and decreasing in size again from the proxi- mal to the distal tip of the smaller sac. Unarmed accessory cirrus as an elongated sac. The larger distal hook is slight- ly curved, 111.5 um long and +43.5 um wide at its base; its base is provided with a slightly curved, +42.3 um-long projection with a blunt distal tip forming a ~90° angle with the axis of the hook. The smaller hook is funnel-shaped, +58.9 um long and +46.6 um wide at its base. Description. General morphology. Live mature spec- imens are 1500-1800 um long (n = 2), with two eyes (Fig. 3A, B). The proboscis is 297-304 um (n = 2) long in swimming animals and is characteristic for koinocyst- idids (Brunet 1972; Karling 1980; Diez et al. 2021). The pharynx (ph, Fig. 3A, B) is positioned near the body’s midpoint and has an approximate diameter of 231- 258 um (n = 2) in the live specimens. The oesophagus is visible as a clear zone surrounded by oesophageal glands behind the pharynx. It empties into the intestine, which is situated in the posterior portion of the body. The male and female reproductive systems are mainly located in the third posterior region of the body. zse.pensoft.net 884 Tsuyuki, A. et al.: Two new species of Koinocystididae, Rhabdocoela, from Japan 50 um (F) Figure 3. Reinhardorhynchus sagamianus n. sp., NSMT-DNA 56985 (paratype) (A) and NSMT-PI 6460 (holotype) (B-H). A, B. Micro- graph and drawing of live animals. C. Detail of the male copulatory organ in a live animal. D, E. Drawing and micrograph of the belt of overlapping spines in the armed cirrus of the male copulatory organ in a whole mounted specimen fixed in Entellan New. F—H. Draw- ings and micrograph of the distal hooks associated with the male copulatory organ in a whole mounted specimen fixed in Entellan New. zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 877-895 Male reproductive system. Paired testes anterior to the pharynx are on each side of the body (t, Fig. 3A, B). A pair of sac-like seminal vesicles (sv, Fig. 3B—D) fuse distally before entering the copulatory organ and forming an ejacu- latory duct (ed; Fig. 3C, D). The piriform copulatory organ is 323—376 um (n = 2) long and encompasses a proximal prostate vesicle, a bipartite armed cirrus, and an unarmed accessory cirrus. Distally, it bears two large hooks of vary- ing shapes (hl, h2, Fig. 3D). The prostate vesicle consists of one type of intracapsular prostate gland (pg1; Fig. 3B— D) opening into the transition zone between the ejaculatory duct and armed cirrus through filiform ducts (Fig. 3B—D; Suppl. material 1). A second type of extracapsular prostate gland (pg2; Fig. 3B—D) enters the copulatory organ prox- imally. The armed cirrus bears small spines on its entire surface (cia, Fig. 3B—D; Suppl. materials 1, 2) and has two sacs of differing sizes, both of which are equipped with an interconnecting belt of overlapping, scale-like spines (Fig. 3B-F; Suppl. material 1). The spines of the proximal part of the spiny belt start in the larger sac. Here, the scale-like spines are triangular, more spaced out, and 20.1 um long (cps, Fig. 3B—F). Gradually, these spines become less trian- gular, more overlapping, and decrease in size (8.8 um long) until they reach the transition to the smaller sac, where they fold backwards and continue into the smaller sac (cms, Fig. 3C, E, F; Suppl. materials 1-3). From this fold, the spines gradually increase in size (from 6.1 to 22.2 um long) to- wards the proximal tip of the smaller sac, where they fold again to continue along the distal side of this sac while gradually decreasing in size again (cds, Fig. 3C, E, F). The total length of this spiny belt is 295.2 um. The unarmed accessory cirrus (clu, Fig. 3C, D; Suppl. material 1) runs alongside the armed cirrus from the proximal end of the copulatory organ to the distal end of the armed cirrus. It appears as an elongated sac that narrows proximally. It is possible that this narrow proximal part of the unarmed accessory cirrus connects to the bundles of extracapsular prostate glands (pg2, Fig. 3C) visible around the copula- tory organ and seminal vesicles, but this connection could not be established with certainty. The larger distal hook is 111.5 um long and 43.5 um wide at its base (h1, Fig. 3G, H; Suppl. material 1). One side of the base features a sturdy, slightly curved projection with a blunt distal tip. The pro- jection itself is 42.3 um long and forms an ~90° angle with the axis of the hook. The smaller distal hook is 58.9 um long and has a 46.6 um-wide base (h2, Fig. 3G, H). Female reproductive system. The vitellaria (v1; Fig. 3A) extends from the rear end of the pharynx to the pos- terior body end. Paired ovaries with oocytes arranged in a single line are situated anterior to the copulatory organ (ov, Fig. 3B, C). The bursa (bu, Fig. 3B) opens into the female atrium through a muscular bursal stalk (bs, Fig. 3B). The female atrium also receives the female duct (fd, Fig. 3B), which consists of a narrow distal duct in which bundles of female glands (fg, Fig. 3B) discharge, and a wide, muscular proximal part that receives the oviducts. Etymology. The species epithet sagamianus refers to the type locality, which is located in Sagami Bay. Distribution. Kanawaga, Sangashita Beach, Japan. 885 Molecular phylogeny The resulting ML and BI trees were congruent with each other in terms of topology, so only the ML tree is shown in Fig. 4. All of the examined koinocystidid species form a clade with full support. Within this clade, four major clades can be recognized: (1) a clade composed of Utelga heinckei (Attems, 1897), Utelga pseudoheinckei Karling, 1980, and Parautelga sp. (with full support); (2) a clade with Koinogladius sinensis (Wang & Lin, 2017) and Rhinolasius dillonicus Karling, 1980 (‘sinensis’ clade in Diez et al. (2021)) (PP = 1.00; SH-aLRT = 99.5%; UF- Boot = 99%), (3) a clade composed of four representa- tives of /taipusa (‘divae’ clade in Diez et al. (2021)) (with full support), and (4) a clade with several representatives of Reinhardorhynchus, Itaipusa sp. 1, and Koinocysti- didae sp. 1 (‘rieger?’ clade in Diez et al. (2021)) (PP = 0.82; SH-aLRT = 99.1%; UFBoot = 82%). Our two new species, R. ryukyuensis sp. nov. and R. sagamianus sp. nov., from Japan, are nested in the ‘riegeri’ clade. Rein- hardorhynchus ryukyuensis sp. nov. is the sister taxon to a clade including R. hexacornutus Jouk, Diez, Reygel & Artois, 2021, R. tahitiensis Jouk, Diez, Yurduseven, Rey- gel & Artois, 2021, and an unidentified species of Koino- cystididae, albeit with relatively low support (PP = 0.75; SH-aLRT = 99.3%; UFBoot = 54%). Reinhardorhynchus Sagamianus sp. nov. is sister to a clade consisting of R. riegeri (Karling, 1978), R. anamariae Diez, Reygel & Artois, 2021, and /taipusa sp. 1 with full support (Fig. 4). Faunistic account A total of 58 taxa of marine and brackish water microtur- bellarians that have been identified to species level have been recorded from the coastal areas of Japan, including the two new species of Reinhardorhynchus described in this study; four taxa were only identified up to genus level (Table 3). All these taxa belong to the Macrosto- morpha, Rhabdocoela, Prolecithophora, and Proseriata. Nineteen species were found in brackish water habitats, all of which belong to genera that are either considered typical to these environments or euryhaline marine taxa. Most of the records are from Hokkaido (33 taxa) and the northern part of Honshu (18 taxa) (Table 3; Fig. 1). A few records are from locations in southern Honshu (Kanagawa, Okayama, and Shimane) and the Ryukyu Islands (Okinawa). Discussion Morphology Koinocystididae is one of the most species-rich groups of kalyptorhynch rhabdocoels. Its representatives are found globally in marine sediments and on seaweeds; however, some species also occur in freshwater habitats. Most koi- nocystidids have a large proboscis with a sphincter at the zse.pensoft.net 886 | Reinhardorhynchus ryukyuensis n. sp. ' Reinhardorhynchus ryukyuensis n. sp. Reinhardorhynchus sagamianus N. sp. Reinhardorhynchus riegeri 1.00/99.3/100 Reinhardorhynchus riegeri 1.00/82,7/76 0.75/99.3/54 1.00/99.8/100 0.73/100/50 0.82/99.1/82 0.73/96.7/50 0.04 -/11.2/32 e Reinhardorhynchus hexacornutus Reinhardorhynchus tahitiensis B [taipusa sp. 1 Tsuyuki, A. et al.: Two new species of Koinocystididae, Rhabdocoela, from Japan Reinhardorhynchus tahitiensis A Koinocystididae sp.1 Reinhardorhynchus anamaeriae Sekerana stolci e | ' /taipusa divae LeeLee karlingi 1.00/98/97 1.00/94.8/97 0.75/93.9/46 e 0.59/78/41 0.99/91 oe e 0.73/94.7/56 | 1.00/99.5/99 | Koinogladius sinensis YTP 1 Koinogladius sinensis YTP 3 Koinogladius sinensis YTP 2 Rhinolasius dillonicus Utelga pseudoheinckei r /taipusa novacaledonica Itaipusa biglandula Utelga heinckei Utelga heinckei e Utelga heinckei | Utelga heinckei 1.00/81.9/77 Parautelga sp. Mesorhynchus terminostylis [———_ Cystiplex axi e -—_______—_ Cystiplex sp. | Outgroup Figure 4. ML phylogenetic genetic tree based on a concatenated dataset of partial 18S and 28S rDNA sequences. Branch support values are indicated next to the nodes as posterior possibilities/SH-aLRT values/UFBoot values. Black dots indicate the maximum values for all support measures. base of the cone and complex atrial organs with sclerotized structures and a copulatory bursa (Brunet 1972; Karling 1980; Diez et al. 2021). The two new koinocystidid spe- cies from Japan exhibit the characteristics described for the recently proposed genus Reinhardorhynchus. At pres- ent, this genus accommodates sixteen species, half of which were newly described by Diez et al. (2021). Repre- sentatives of Reinhardorhynchus all have large accessory hooks or spikes associated with the male copulatory organ, in contrast to species of /taipusa, which lack such accesso- ry structures (Karling 1978, 1980; Diez et al. 2021). The new species of Reinhardorhynchus from Okinawa, R. ryukyuensis sp. nov., has a unique combination of fea- tures. Firstly, R. ryukyuensis sp. nov. has no armed cirrus in its male copulatory organ. However, the presence of a short, unarmed cirrus cannot be excluded, as the exact ending of the prostate glands and ejaculatory duct in the tip of the penis papilla could not be observed. In all other known species of Reinhardorhynchus, except for R. scoti- cus (Karling, 1954), the male copulatory organ possesses zse.pensoft.net one (R. hexacornutus Jouk, Diez, Reygel & Artois, 2021, R. renei (Reygel, Willems & Artois, 2011)), two (R. rieg- eri (Karling, 1978), R. unicornis Diez, Aguirre, Reygel, & Artois, 2021, R. variodentatus (Karling, Mack-Fira, & Doerjes, 1972)) or three (R. pacificus Diez, Reygel & Ar- tois, 2021) armed cirri provided with fields of spines, or a single cirrus armed with belts/rows of overlapping spines (R. anamariae Diez, Reygel & Artois, 2021, R. beatrizae Diez, Aguirre, Reygel & Artois, 2021, R. bispina (Karling, 1980), R. curvicirrus (Karling, 1980), R. evelinae (Mar- cus, 1954), R. riae Diez, Reygel & Artois, 2021, R. ruffin- Jonesi (Karling, 1978), R. soror Diez, Reygel & Artois, 2021, R. tahitiensis Jouk, Diez, Yurduseven, Reygel, & Artois, 2021) (Karling et al. 1972; Karling 1980; Rey- gel et al. 2011; Diez et al. 2021). Reinhardorhynchus ryukyuensis sp. nov. differs from R. scoticus because it has a girdle of five smaller interconnected hooks around the distal tip of the penis papilla rather than a circular plate with fine needle-like spines as in R. scoticus, and because it has two larger separate hooks instead of three to five Zoosyst. Evol. 100 (3) 2024, 877-895 foldable hooks at the base of the penis papilla (Karling 1954, 1963; Ax 2008). Secondly, the configuration of the larger and smaller hooks in R. ryukyuensis sp. nov. mark- edly deviates from the sclerotized hooks and spines found in other species of Reinhardorhynchus. The complex ar- rangement of the five smaller hooks on a girdle composed of two semi-elliptical plates surrounding the penis papilla is unique among representatives of the genus. Another species, R. riae, also has sclerotized structures associat- ed with a pseudocuticular penis papilla, but in this spe- cies, these structures consist of two flattened hooks with a broad and rounded distal end (Diez et al. 2021). In ad- dition, R. riae also has a cirrus armed with two spinous rows. The above-mentioned differences make it easy to distinguish R. ryukyuensis sp. nov. from its congeners and thus warrant the description of a new species. The new species of Reinhardorhynchus from Kanaga- wa, R. sagamianus sp. nov., 1S also unique among its con- geners because it is the only species with the combination of a bipartite armed cirrus with a belt of spines, an un- armed accessory cirrus, and two distal accessory hooks associated with the male copulatory organ. Only three other species of Reinhardorhynchus, R. riegeri, R. unicor- nis, and R. variodentatus, possess two cirri and one (R. unicornis) or two (R. riegeri and R. variodentatus) acces- sory hooks in their male copulatory organ. However, in these three species, both the cirri are provided with spines. Eight other species of Reinhardorhynchus, R. anamariae, R. beatrizae, R. bispina, R. curvicirrus, R. riae, R. ruffin- jonesi, R. soror, and R. tahitiensis, possess a single cirrus armed with belts or rows of spines and two distal hooks (Karling 1978, 1980; Diez et al. 2021). One species, R. evelinae, has a single cirrus with hooked denticles and two or three rows of spines in combination with three distal spines (Marcus 1954). Apart from the lack of an unarmed accessory cirrus in all these species, other noticeable dif- ferences in the morphology of the male copulatory organ are present when compared to R. sagamianus sp. nov. The two distal hooks in R. riae and R. tahitiensis are symmet- rical and have broad and rounded distal ends. In addition, these two species have multiple spinous belts — two in R. riae and four in R. tahitiensis (Diez et al. 2021) — and are, therefore, very distinct from R. sagamianus sp. nov. and the other six species mentioned above. The morphology of the spiny cirrus belt, including its overall length and shape and the shape and size of the individual spines, is unique in every species of Reinhardorhynchus with such a belt. In R. bispina, these spines are mostly uniform in size, whereas in R. anamariae, R. beatrizae, R. curvicirrus, R. ruffinjonesi, R. sagamianus sp. nov., and R. soror, there are clear sections with a gradual increase or decrease in the size of the spines (Karling 1978, 1980; Diez et al. 2021). In R. soror and R. ruffinjonesi, a row of larger hook- or claw-shaped spines is also present in the belt (Diez et al. 2021). Another unique feature of the new species from Kanagawa is the fact that the armed cirrus has two sacs, which results in the unique curved and twisted contour of the spinous belt as it spans the different compartments of 887 the cirrus. While described as a “fold in the cirrus wall,” the configuration of the armed cirrus in R. curvicirrus also implies the presence of a blind sac (Karling 1980). A small “spiny diverticulum” as part of the armed cirrus has also been described for R. ruffinjonesi (Karling 1978; Diez et al. 2021). Finally, only R. beatrizae and R. soror share the presence of a projection on the base of one of the dis- tal hooks with R. sagamianus sp. nov. In R. sagamianus sp. nov., this projection has a blunt, angled distal tip and is oriented at a ~90° angle to the main axis of the larger distal hook. In R. beatrizae, this projection is more fun- nel-shaped, distally more pointed, and at an angle of more than 100° to the main axis of the hook (Diez et al. 2021). In R. soror, the tip of the largest hook is noticeably curved compared to R. sagamianus sp. nov., which has a straight tip. Also, there are two projections on the base of the larg- est hook of R. soror: one long straight, funnel-shaped pro- jection with a blunt tip making an angle of 90° to the main axis of the hook, and one shorter, more or less square and folded projection (Diez et al. 2021). These characteristics are clearly different from R. sagamianus sp. nov. Molecular phylogeny The interrelationships of the Koinocystididae have been extensively discussed by Diez et al. (2021) based on the results of molecular phylogenetic analyses inferred from 18S and 28S rDNA sequences. Our reconstructed tree is congruent with these results and shows that the new spe- cies R. ryukyuensis sp. nov. and R. sagamianus sp. nov. form a clade with five other species of Reinhardorhyn- chus and the unidentified taxa /taipusa sp. 1 and Koino- cystididae sp. 1 with high support (‘rieger7’ clade in Fig. 4). With 12 out of 18 species of Reinhardorhynchus still lacking from the analyses, the molecular phylogenetic interrelationships within the ‘riegeri’ clade are difficult to interpret within the context of character evolution. Re- inhardorhynchus ryukyuensis sp. nov., R. hexacornutus, and R. tahitiensis belong to the same clade and have a clearly different morphology from other species of Re- inhardorhynchus. For example, the lack of an armed cir- rus, the presence of two large separate hooks, and a distal girdle of five smaller hooks in the male copulatory organ are unique features of R. ryukyuensis sp. nov. However, it does share this relatively large number of hooks with R. hexacornutus, which has six separate hooks associat- ed with the male copulatory organ; moreover, these six hooks are also organized in two groups (4+2) (Diez et al. 2021). In all other species of Reinhardorhynchus, the number of hooks or spines not associated with a cirrus is either one, two, or three, except for R. scoticus, which has five separate sclerotized structures associated with its copulatory organ. The male copulatory organ of R. tahi- tiensis has an armed cirrus with four rows of spines that vary in size and two similar, blunt accessory hooks. Rein- hardorhynchus sagamianus sp. nov. forms a clade with R. anamariae, R. riegeri, and Itaipusa sp. 1. 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Evol. 100 (3) 2024, 877-895 eMeUlyO pue|s| Iyesiys| (Z lysayyy ‘einqy ‘UBJOINIA] ‘PUe|S] WIYSIY ‘pue|s| = (41861) exleL (Z ‘OpleyyOH(T unqay ‘eewewWo] ‘OJOYsQ (T (W) (T86T ‘eylleL) feznuiy eloonuueropnasd pue|s] UIYSNYO = (8Z6T) exilel opleyyOH ‘Iysayyy ‘ayepoyey ‘o10YsC (W) SZ6T ‘exlfel epe4.so011q e1odouAsoja09 (996T Sulvey) YSN ‘elUsoyleD “(Z86T SJa|Y4-Hodos pue siajyz ‘6/61 S4ajys-0dos pue xy) nines ‘lewoqey ‘Owly YSN ‘UO}sulyseM -(O66T SelUOWY pue xy) YSN ‘exsely (41861) eller opley4OH ‘Wyse Mey ‘UeyIYs| ‘IYsSeyy (W) (996T ‘Sulldey) epiusesed epsnuanu| leWOqeH mi (O86T) exifeL opleyxOH ‘1ysisiey ‘nddeyly aded ‘o10ysQ (IW) OS6T ‘eylleL evesiuuld euozz Fi (SZ6T) exileL opleyyOH lysayyy ‘a}epoyey ‘OJOYSC (W) SZ6T ‘exllel eyounsuod esodouAsoj909 5 (O86T) exieL opleyxOH lewoqey (W) O86T ‘exlfeL sisualewiogey euozy aepluodouAso0j909 eMeULYO puels] eluNsayeH (€ ‘pue|s} (€ ‘emeseuey euwlys-esor (Zz ‘ulyseqy ‘eyeply ig (27861) exifel (Z OpleyyOH(T —_‘e|NSuulUag epawey ‘Iysisiey (T (W) Z86T ‘exile, aeooesew euejdozyz eMeUIYO Spue|s| ewessey - (27861) exIeL (Z -OpleyyOH(T (Z slyoeynsey ‘OWA aded (T (IW) Z86T ‘eyllel euosAxo euejdozz aepiue|doyewan a (8002) XV lJOWOY puod 1yonqo (G) 8002 “XV e2nuiW eUuoUI) ia (8002) XV lJOWOY JONY ASeYe| ‘JAAIY |EYON (@) 8002 ‘XY eo/uode/ eul/IYoy iz (8002) XV lJOWOY aye] uesne (g) 8002 ‘xy ed/uode! euoulwojdnq = (8002) XV |JOWOY JaAy aseyel (@) 8002 ‘XY siuopIy UOUIWOjdng = (8002) XV opley4OH OANWAN “EWOGeH “UeJOINIA| (W) 2861 ‘exeL e/naisanoyoijop CUoUI| nines ‘ulyseqy ‘leWwoqeH “lYyseyxy4y - (42861) exlleL ople44OH ‘nsyeqnyeys ‘eyngy ‘OJ0YysC (W) Z86T ‘exIeL s/o1/dl4Z0d/09 sIja20u0/) = (4Z86T) exlleL opley4OH OJOUSO (W) Z86T ‘eyllel erluode! ejjaua} sja90u0/) = (42861) ewleL opley4OH lusayyy ‘NS}equo (WW) (Z86T ‘exifel) aeyn! eulyile] a (42861) eel opley4OH euejas ‘1ysisiey ‘IOowny (W) Z86T ‘eyilel si/eulseaiajad euoul SePIpl|sOUuo|| Bye119SO1d = (8002 -66T) XV lJOWOY JOAY BSCE] JOAY OJLUILWOY (€) P66T ‘XW enjosu! euejdoueder JaAly aseyel] ‘puog oyoyeyel (LZ6T AouoseN) elssny ‘JeAsod (8002) XV lJOWOY JOAY OJEUIWOY “aye7] UeSNE (d) L261 ‘AouoseN OYY eelUadA/Ny aepneiuadninin o (0202) !wo SUPLUIYS luineyen ‘aye IulyS (G) OZOZ “WO esungeuissisuap &/NWO}SOs9,UF ‘(8Z6T Sulyey) epnulag ‘(Z96T Sulvey) YSN “eloped “(ZZ6T ‘Ie 39 suley) VSN ‘WeMeH “(TGET Snovey\) |IZe1g ‘oenseqas ORS AP Pull (GGET Pe|AISAM) WN “UOAE -(ETET J4e49) aUles¥N) ‘|Odoysenss ‘(QO6T Auoye7-seuly ‘ETE T ‘7881 (sdjay (IW) SG6T ‘Peiqisem }J219) Ajey ‘eas IeUPY <(6G6T xy) Aeyn] ‘epeljeuly (8102) |wO opley4OH W044) PUR|S| LIYSIY ‘eUEYYeN\ (Z88T ‘JesD) WNYIO/JOUOW PLIO}SOLPUI|AD (SS6I1 pe|qysam) POD ‘eLAS| ‘(GGET PelqiseaM ‘Ov6T sulyey) AKeMJON ‘puelsaa ‘(GGET pelqisem) uapams ‘usewyin5 (ZG6T Pelqisem) MN ‘spue|s| pue|yle4 -(ETET Je49) aulesyf) ‘|Odoysenas (ETET Jes) Aled ‘ase (ETST HEI) YN) “YINOWAld (GGET PeIGISAM “ETET HEAD) WN ‘uel 0 AIS] (Q6QT UUeWUYN4) soUeI4 ‘NeauiedU0D (0ZOZ) WO ‘(GGET) peiqisan emeseuey IyeSl|N (IN) (96QT UUeWYyN4) Wninp ewojsol|yi eepiwoysopnesd (IN) 6261 ic (8102) !wo ople44OH pues} UIYSIY ‘youuliny asua/Ny WNjeqo] WINWO}SO/se|d ewekeyo - (QT6T) emezo] (Zz ‘emeseuey( T Opewlysy (Z {yesI| (T (IN) SI6T ‘emezol few SosaoILI0A SePIWOSOIse|d es0ydoyyda|O1dg ueder ueder jo apis}no uonnqiisig adUdI9JOyY ueder ul Ayjeo07 AyUSp! D1WOUOXeL Ul 8.4N}99Ja1g zse.pensoft.net (4,9°SZ,0S 0221 ‘(ZZOZ) ‘|e }8 pueig Aq Sipas aeyaoul Se pasapisuod AfuawNd SI eUNgpelg snuas aus “elssny pue ueder yjog Aq pauleo si pues) slyseuny}; ‘PalapIsSuOd OS|e SEM SQ6T ‘Buley JUIUOPAS *D YBnouye ‘TZOZ ‘SloLUy * aysiyusaIS UeA ‘JaBAay ‘W4aqoy s/sual/emeY “D Ajayi] ISOW ‘eUe/dI/aYD JO Saldads JUaalJIp & 0} Buojaq 0} Ueder WO SUBWIDadS BU} PasapISUOD (TZQZ) ‘Je 18 Yaqod, ‘gunjoajaid yoea Ul peusISse SJBQLUNU aU) 0} PUOdsau0d AjeD0] YDeA UI SaquUNU ay] Tsuyuki, A. et al.: Two new species of Koinocystididae, Rhabdocoela, from Japan (EZOZ) ‘Ie 38 aYsIyUaa}S Ue) eMeUlyO ‘N.1'G0,62.92) CULO (IW) “ds snyouAysoposeyried aeplyoUAYsOZIYIS (4.922.290.2271 (EZOZ) ‘Ie 38 aYsIyUaa}S UA eMeulyO ‘N.6°L 1,270.92) 123] (IN) “ds esjaznesed aepipysAsouloy ejaooopqeyy (,9Z°00,0.ZET ‘N.60°6T,Z TLE) (6002) MlyseAeqoy eMEYIYS| eiINSUUIUa, OJON ‘YOeag IwWeuey (I) ‘dS Winwojsolep (4.097 L.Ov.6€T ‘N.v2' TE,80.G€) (6002) !yseAeqoy emeseuey eINSUUIUD INIA] ‘Og eAeley (IN) sds eungpeig SePIWO}sO1Ie | PYCIOWO SOE saldads payljuapiun (W) 1Z0¢ ‘Heeyx20Y9S = (21861) exifel opley4OH (O40YSQ) OJNGeyo}eS ‘oyngeyIy9A, §—-*B HAHEH-1uUND (TY6T ‘eyifeL) 107! syjaoeyifel aepipla.ouOWIYoAy = (6Z6T) exieL opley4OH eAOL (W) 6261 ‘exile ezeauljojjnd euejdojewan) = (6Z6T) exieL opley4OH lysisiey (W) 6Z6T ‘exIeL ae/naisanol/io eue/dojewan eepiue|doyewan) = (786T) exieL opleyyOH lysisiey ‘Ue JOIN] (W) V86T ‘exIeL evapi/7so/0eW e//aidsey] emes ‘iyseqy ‘TEWOeH ‘Iysayyy ‘IUeLUeS (W) €86T - (2C€86T) ewer opleyyOH ‘UBJOINIA| ‘IUSIBIeY ‘WeYIUS| ‘eylle) SuejnoljofoulMas euej;doAleo0j0N 5 (9€86 1) exifeL opleyxOH UBJOINIA (W) E861 ‘exlleL ezesopad euejdopqeysAjod fe (GE86T) eleL opley4OH eyIYS| (W) E861 ‘exWfeL /epewed euejdozoyoiy a (GE86T) eleL opley4OH LJe4IYS| (W) €86T ‘exlleL sysusoze euejdo}0sh7 = (GE86T) exlleL opley4OH eqnqy (W) E86T ‘exIfeL sisuaeznge eue/dojoyay aepiue|doio 1yoeyney . (41861) eel opley4OH ‘UeJOIN\| ‘pue|s| LIYSNYO (W) T86T ‘exlleL ereye esodouAsoja05 owl ade) a (ZZ6T) exileL opleyxOH ‘pue|s| ungay ‘Iyseyyy ‘O10YSC (W) ZZ6T ‘exllel esoyjidediy ersonuue, aeplodouAso0j909 B}ELaSO1d ueder ueder jo apis}no uolnnqiysig a0Ud19JOy ueder ul Ayje207 AyUSp! D1WOUOXeL 890 ul 8.4N\99Je1g zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 877-895 species in this clade all have the combination of at least one armed cirrus and two large, heteromorphic accesso- ry hooks (Diez et al. 2021; this study). In addition, R. sagamianus sp. nov. and R. anamariae both have a con- spicuous belt consisting of overlapping lamellar spines of varying sizes in the armed cirrus. It 1s possible that other species of Reinhardorhynchus with such a combination of characters, including R. beatrizae, R. curvicirrus, R. ruffinjonesi, and R. soror, also gather in this clade, but further analyses with more dense taxon sampling are needed to confirm its synapomorphic traits. Marine microturbellarians in Japan Most of the species recorded in Japan have been accurate- ly identified except for a prolecithophoran that most like- ly belongs to the species A//lostoma durum (Fuhrmann, 1896) and four unidentified species of Macrostomorpha (Bradburia, Macrostomum) and Rhabdocoela (Carcharo- dorhynchus, Parautelga), respectively (Table 3). The pu- tative representative of A//ostoma durum lacks a specified collection locality in Japan based on our literature survey (Westblad 1955; Omi 2020). The four unidentified spe- cies await further description. Some microturbellarians found in Japan, including specimens likely belonging to Plagiostomidae (Vorticeros, unidentified genus) and Cy- lindrostomidae (unidentified genus) collected from Rishi- ri Island (Hokkaido), are not listed in Table 3 because the individuals were immature (Omi 2018). Resampling will be required for an accurate identification. Macrostomorphs, rhabdocoels, prolecithophorans, and proseriates are the most commonly encountered micro- turbellarians in marine and brackish water environments around the world. It is, therefore, not surprising that all microturbellarians collected in Japan so far belong to these groups (Table 3). Other marine taxa of free-living microturbellarians, including catenulids and gnosonesim- ids, are rarely encountered, and while they seem to have widespread distributions, they have not yet been found in Japan. Most recorded species were collected in Hokkaido and northern Honshu as a result of the research activities conducted in these areas over the years (Evdonin 1969; Tajika 1978, 1981b, 1982a, 1982b, 1983b, 1983c; Ax 1994, 2008; Omi 2018; Takeda and Kajihara 2018; Van Steenkiste et al. 2023). Japan extends from 20° to 45° north latitude and consists of more than 14,000 islands and almost 30,000 km of coastline. This results in a wide variety of marine habitats and climatic conditions, from seasonal sea ice along the northern coasts of Hokkaido to tropical coral reefs around the atolls and islands of the Ryukyu and Ogasawara Islands. Undoubtedly, rich com- munities of microturbellarians are also present in these diverse but unexplored marine areas of Japan. Only nine out of 58 species of marine or brackish wa- ter microturbellarians from Japan have also been collect- ed in other parts of the world (Table 3). Our overview indicates that some of these species might be confined 891 to the regional seas around Japan (Palladia nigrescens (Evdonin, 1971), Multipeniata kho Nasonov, 1927) or the Northern Pacific (Pitychopera japonica Ax, 2008, In- venusta paracnida (Karling, 1966)), while others have widespread distributions (Cheliplana setosa Evdonin, 1971, Cheliplana terminalis Brunet, 1968, Trigonos- tomum vanmecheleni Artois, Schockaert, Beenaerts & Reygel, 2013, A/lostoma durum (Fuhrmann, 1896), Cy- lindrostoma monotrochum (von Graff, 1882)). The fact that 49 species have only been recorded from Japan does not necessarily indicate a high degree of endemism for microturbellarians in the marine areas of Japan, but rather exemplifies how little species discovery and exploration has been done in the surrounding coastal areas of the Rus- sian Far East, the Korean peninsula, Eastern China, and the Philippines. The scarcity of researchers focusing on various meio- faunal groups, such as microturbellarians, has been rec- ognized as a significant challenge that needs urgent atten- tion (Schockaert et al. 2008; Balsamo et al. 2020). Marine microturbellarians play crucial roles in the trophic dy- namics of coastal marine environments (Urban-Malinga 2011; Leasi et al. 2016, 2018; Schratzberger and Ingels 2018; Martinez et al. 2019; Balsamo et al. 2020). Hence, understanding their diversity and interactions with other organisms, including prokaryotes, protists, and other mi- cro-invertebrates, 1s essential for evaluating their impact on marine ecosystems along the Japanese coasts. Micro- turbellarians also establish symbiotic relationships with other micro-organisms. The rhabdocoel representatives of Pogaina, of which two species have been recorded from Japan (Table 3), are known to practice kleptoplasty by se- questering plastids from diatom prey cells (Van Steenkiste et al. 2019). Recent studies identified single-celled para- sites in microturbellarians from Japan and other parts of the world as apicomplexans belonging to the genus Rhyt- idocystis (Holt et al. 2022; Van Steenkiste et al. 2023). An apicomplexan cell was also observed inside the intestine of R. ryukyuensis sp. nov., which was collected in the same location as Carcharodorhynchus sp. (Table 3), one of the host taxa in the study of Van Steenkiste et al. (2023). It is therefore likely that the apicomplexan in R. ryukyuensis sp. nov. also belongs to the genus Rhytidocystis. The ef- fects of these interactions on the flatworm hosts and their symbionts or parasites are still largely unknown. Under- standing these complex ecological relationships is crucial for addressing the challenges in environmental manage- ment and conservation in the region (Zeppilli et al. 2015). 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Van Steenkiste Data type: mov Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.100.120244 suppll Supplementary material 2 Reinhardorhynchus sagamianus sp. nov — detail of the male copulatory organ in a live animal Authors: Aoi Tsuyuki, Jhoe Reyes, Yuki Oya, Kevin C. Wakeman, Brian S. Leander, Niels W. L. Van Steenkiste Data type: mov Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zse.100.120244 suppl2 Supplementary material 3 Reinhardorhynchus sagamianus sp. nov — detail of the male copulatory organ in a live animal Authors: Aoi Tsuyuki, Jhoe Reyes, Yuki Oya, Kevin C. Wakeman, Brian S. Leander, Niels W. L. Van Steenkiste Data type: mov Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zse.100.120244 suppl3 zse.pensoft.net