Zoosyst. Evol. 100 (2) 2024, 437-445 | DOI 10.3897/zse.100.114482 > PENSUFT. Arte BERLIN Description of a new marine flatworm of Prosthiostomum (Platyhelminthes, Polycladida, Prosthiostomidae) from the South China Sea Hai-Long Liu!*, Da-Hao Lin’, An-Tai Wang!, Zhang-Li Hut, Yu Zhang? 1 Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, Guangdong Engineering Research Center for Marine Algal Biotechnology, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, China 2 Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen, China https://zoobank. org/47BB271B-27F 7-48 E6-A94B-C5 16DAEE2B53 Corresponding author: Yu Zhang (biozy@szu.edu.cn) Academic editor: Pavel Stoev # Received 22 October 2023 # Accepted 14 March 2024 Published 12 April 2024 Abstract A new species of the polyclad genus Prosthiostomum is described from the intertidal zone of the South China Sea, Huidong, China, based on morphological and molecular analyses. Prosthiostomum huidongense sp. nov. is characterized by 1) few marginal eyes scattered between the marginal band and the cerebral eyes; 11) sucker located at two-thirds of the body length, being removed from the female gonopore by twice the distance between the male and female gonopores; ii1) shallow male atrium with slightly ruffled inner wall, positioned approximately perpendicular to the body wall. Molecular phylogenetic analyses based on 28S rDNA sequence showed that the new species was nested in a clade composed of Prosthiostomum species. The uncorrected p-distance of COI be- tween P. huidongense sp. nov. and other Prosthiostomum species ranged from 20.3 to 24.3%, and the high genetic divergence further supports P. huidongense as a new species. Key Words Cotylea, genetic distance, molecular phylogeny, morphology, taxonomy Introduction Polyclads are free-living, almost exclusively marine flat- worms with an extremely ramified intestine. They inhabit a variety of environments ranging from the intertidal zone to the deep-sea, such as rocky shores, sand/mud flats, coral reefs, and deep-sea hydrothermal vents (Newman and Can- non 2003; Wolff 2005; Quiroga et al. 2006). Polyclads are important predators in hard bottom environments (Rawlin- son et al. 2011), and prey on crustaceans, ascidians, cni- darians, gastropods, and bivalves (Jennings 1957; Newman and Cannon 2003; Lee 2006; Barton et al. 2020; Teng et al. 2022). Some species feed on scallops and oysters (Newman et al. 1993; Heasman et al. 1998; Gutiérrez et al. 2023), damaging commercial shellfish farming (Sluys et al. 2005). About 1000 species of Polycladida have been de- scribed worldwide; they are classified into two suborders, namely Cotylea and Acotylea, on the basis of the presence or absence of a ventral sucker (Faubel 1983, 1984; Prud- hoe 1985). The cotylean polyclad genus Prosthiostomum Quatrefages, 1845 includes the largest number of species in the family Prosthiostomidae Lang, 1884. It currently contains 54 species distributed worldwide (Tsuyuki et al. 2019, 2021), which are characterized by 1) a pair of free prostatic vesicles without a muscular envelope, 11) a main intestine accompanied by a frontal branch, and 111) a pe- nis armed with a pointed tubular stylet (Faubel 1984). To date, four species of Prosthiostomum have been reported from Hong Kong and Taiwan, China: P. obscurum Stimp- son, 1855, P. grande Stimpson, 1857, P. tenebrosum Copyright Liu, H.-L. 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. 438 Stimpson, 1857 and P. formosum Kato, 1943. Stimpson’s original descriptions were simple and incomplete, lack- ing illustrations. With the exception of P grande, the above-mentioned three species have never been recorded again, and Stimpson’s materials were destroyed during the Great Chicago Fire in 1871 (see Tsuyuki et al. 2021). Therefore, these species remain questionable and cannot be reidentified (cf. Lang 1884). In the last 80 years, since Kato’s (1943) research, species of Prosthiostomum have not been reported from Chinese waters. In this paper, we describe a new species of Prosthiostomum from the coast of the South China Sea based on morphological and mo- lecular data. We selected 28S rDNA for a phylogenetic analysis, considering the fact that this sequence is avail- able for most species of prosthiostomids. Materials and methods Sample collection and morphological studies Three specimens were collected under rocks at the in- tertidal zone in Huidong, Guangdong Province, China (Fig. 1). The worms were measured and then photo- graphed alive with a digital camera. Two specimens were fixed for histological examination following the method modified from Newman and Cannon’s (1995), in which 1) worms are placed onto filter paper, which 1s then placed onto frozen 10% formalin seawater, i1) an additional Vietnam ~“ v3 - = 110 °E Liu, H.-L. et al.: Prosthiostomum from South China sea fixative is added to just cover the worm, then worms are smoothened with a soft brush to ensure they are fixed flat. For histological examination, specimens were dehydrat- ed in an ethanol series and cleared in xylene, thereafter, embedded in paraffin wax. Serial sections were cut at intervals of 7 um and were stained with modified Ca- son’s Mallory-Heidenhain stain solution (see Yang et al. 2020). One specimen was fixed in 95% ethanol for DNA extraction. Histological preparations are deposited at the Marine Biological Museum, Chinese Academy of Sci- ences (MBMCAS), Qingdao, China. Molecular analyses Total DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Germany). Three markers (partial nuclear 28S rDNA, mitochondrial 16S rRNA, and cyto- chrome c oxidase subunit I (COI) sequences) were am- plified by polymerase chain reaction (PCR). The PCR was carried out using the primers: Acotylea_COI F and Acotylea COI R (Oya and Kayjihara 2017) for COI; l6sar-L and 16sbr-H (Palumbi et al. 2002) for 16S; LSU5 and LSU3 (Littlewood 1994) for 28S. Thermal cycling was initiated with 3 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C (COI) or 49 °C (16S) or 52 °C (28S) for 45 s, and extension at 72 °C for 1 min. The cycling ended with a 7-min sequence extension at 72 °C. All amplified 25 °N 20° N 115 °E 120 °E Figure 1. Map showing the sampling site of Prosthiostomum huidongense sp. nov. and photograph of the habitat. zse.pensoft.net Zoosyst. Evol. 100 (2) 2024, 437-445 products were purified using QIA-quick gel purification kit (Qiagen) and sequenced at BGI (Shenzhen, China), by means of double-stranded Sanger sequencing to ver- ify accuracy. Sequences were checked and edited using SeqMan software (DNAStar Inc.). All the new sequenc- es were submitted to GenBank. A total of 27 sequences of 28S rDNA of prosthiosto- mid species were used for molecular analyses; Pseudo- biceros stellae Newman & Cannon, 1994 was selected as outgroup (Suppl. material 1: table S1). Alignments were performed with MAFFT ver. 7 (Katoh and Standley 2013) using the Auto strategy. Ambiguous regions were removed with the web version of Gblocks ver 0.91b us- ing default parameters (Castresana 2000). The Maximum likelihood (ML) analysis was performed at the CIPRES Science Gateway web with RAxML (Stamatakis 2014) on XSEDE, in which 1000 rapid bootstrap replicates and the GTRGAMMA model were used to evaluate and op- timize the likelihood of the final tree. Bayesian inference (BI) was performed using MrBayes ver 3.2.2 (Ronquist et al. 2012), and the substitution model (GTR + G + I) was selected by MrModeltest ver 2.3 (Nylander 2004) according to the Akaike information criterion. MrBayes was run for 10 million generations with a tree being sam- pled every 1000 generations, with two parallel runs and four independent Markov chains per run and with the first 25% of trees discarded as burn-in. The standard de- viation of the split frequencies (<0.01) was used as the criterion to validate the convergence of the analysis. In addition, we determined cytochrome c oxidase subunit I (COI) sequences for DNA barcoding. COI sequences of the Prosthiostomum species currently available from the GenBank were used for the genetic distances analy- sis (Suppl. material 1: table S1). Uncorrected p-distances of COI were calculated in MEGA ver 10 (Kumar et al. 2018). Voucher specimens of the material that was ana- lyzed molecularly have been deposited at the Shenzhen Key Laboratory of Marine Bioresource and Eco-environ- mental Science. Results Family Prosthiostomidae Lang, 1884 Genus Prosthiostomum Quatrefages, 1845 Prosthiostomum huidongense Liu, sp. nov. https://zoobank. org/9 A6C 8F9B-5664-4D50-806A-A50A4E4D 119E Material examined. Holotype: MBM287880, Huidong, 22°44.95'N, 114°45.05'E, Guangdong Province, China; under rocks at the intertidal zone, 21 April 2023, coll. Hai-Long Liu; sagittal sections on 10 slides, deposited at MBMCAS. Paratypes: MBM287881, sagittal sec- tions on 11 slides, same data as for holotype. Molecular voucher specimens: 20230421A1, material of the follow- ing GenBank accession numbers have been deposited at the Shenzhen Key Laboratory of Marine Bioresource 439 and Eco-environmental Science, GenBank: OR680085 (COT), OR680128 (16S), and OR680129 (28S). Collec- tion data are the same as for the holotype and paratype. Diagnosis. Body oval-elongated; dorsal surface cream-colored with numerous yellowish-brown maculae; a pair of cerebral eyes clusters forming an approximate- ly inverted “V” shape; band of marginal eyes extending backwards to behind brain, few eyes scattered between the marginal band and the cerebral eyes; a pair of pros- tatic vesicles distinctly separated; male atrium shallow, inner wall slightly ruffled; sucker located at two-thirds of body length, removed from the female gonopore by twice the distance between the male and female gonopores. Description. Body oval-elongated with rounded ends, 10.8-12.1 mm long and 5.6-7.0 mm wide at its widest in living state (n = 3) (Fig. 2A). Tentacles absent. Dorsal surface smooth, cream-colored, uniformly covered with numerous yellowish-brown maculae, pharynx slightly bulging (Fig. 2A). Ventral surface translucent, without color pattern. Cerebral region without pigmentation and provided with a pair of clusters of cerebral eyes, located at approximately 1 mm behind the anterior margin, each cluster comprising 15—20 eyes (Fig. 2B, C). About 140 marginal eyes irregularly scattered along the anterior body margin, extending backwards to a level just behind the brain; few eyes scattered between the marginal band and the cerebral eyes (Fig. 2B, C). Ventral eyes not ob- served. Frontal branch of the main intestine extending an- teriorly to the brain (Fig. 3B). Tubular pharynx in a pha- ryngeal pocket, positioned in the anterior half of the body. Mouth opening ventrally, located shortly behind the brain (Fig. 3B). Male and female gonopores closely set at the body center, distance between male and female gonopores being 0.6—0.8 mm; sucker situated 1.4—1.8 mm posterior to the female gonopore, twice distance between the male and female gonopores (Fig. 3A, E). Male copulatory appa- ratus comprising a large seminal vesicle, a pair of prostatic vesicles, and an armed penis papilla, located immediately posterior to the pharyngeal pocket (Fig. 3A, C, E). A pair of spherical prostatic vesicles (0.05—0.07 mm in diame- ter) with a thin muscular wall (0.009-0.012 mm thick), located at both sides of the ejaculatory duct (Fig. 3A, D). Seminal vesicle oval, about 0.2 mm in diameter, with a muscular wall about 0.04 mm thick (Fig. 3A, E). Sper- miducal vesicle separately open into the anterior portion of the seminal vesicle (Fig. 3A, D). Penis papilla armed with pointed tubular stylet (0.14—-0.17 mm in length), enclosed in penis pouch, protruding into male atrium (Fig. 3C). Shallow male atrium with slightly ruffled inner wall, positioned approximately perpendicular to the body wall (Fig. 3A, E). Female reproductive system consisting of vagina, cement pouches, and uteri. Vagina short about 0.1 mm long, lined with a ciliated epithelium; the dorsal portion of the vagina enlarged and anteriorly curved, con- necting to the uteri (Fig. 3A, E). Cement glands numer- ous, concentrated around the vagina (Fig. 3A, E). Pair of oviducts converging before joining proximal end of vagi- na. Ovaries not discerned. zse.pensoft.net 440 Liu, H.-L. et al.: Prosthiostomum from South China sea Figure 2. Prosthiostomum huidongense sp. nov. A. Photograph of entire body, 20230421A1 (left, living specimen in dorsal view), MBM287881 (middle, preserved specimen in dorsal view, paratype), MBM287880 (right, preserved specimen in ventral view, holotype); B. Schematic diagram of head, showing arrangement of the eyes; C. Magnification of head, dorsal view (holotype); D. Magnification of genital pore and sucker (paratype). Abbreviations: fg — female gonopore; mg — male gonopore; ph — pharynx; su — sucker. Scale bars: 1 mm. Etymology. The name of the new species 1s originated from the name of Huidong City, Guangdong Province, China. Distribution. So far only known from Huidong, Guangdong, China. Habitat. Intertidal, under rocks. Molecular phylogeny and genetic distances. The BI and ML trees based on the partial 28S sequences (904 bp) are almost identical in their general topol- ogy, and we show only the ML tree (Fig. 4). In the phylogenetic tree, all Prosthiostomum species form a clade with high support values (BP = 81%, PP = 1). Prosthiostomum huidongense sp. nov. is sister to the clade formed by all other species of the genus, exclud- ing P. lobatum, with high support values (BP = 78%, PP =1). The uncorrected p-distances of the partial COI sequences (658 bp) of the 11 Prosthiostomum species/ molecular entities is 5.7—24.3% (Suppl. material 1: ta- ble S82). zse.pensoft.net Discussion Our specimens are characterized by i) a median frontal branch of the main intestine, 11) a pair of prostatic vesi- cles that are distinctly separated, and 111) a penis armed with a pointed tubular stylet, features consistent with the generic diagnosis of the genus Prosthiostomum as proposed by Faubel (1984). The new species is easi- ly distinguished from the four species reported from China (P. obscurum, P. tenebrosum, P. grande, and P. formosum) by the dorsal coloration and arrangement of the eyes. Among these species, P. obscurum is pale reddish-brown with red spots, a light-colored middle band and a pair of linear clusters of cerebral eyes, each composed of 8-10 eyes; P. tenebrosum is dark gray or sub-black; P. grande has reddish-brown maculae or spots, with a darker longitudinal band in the middle; P. formosum is uniformly chestnut brown without any color pattern. 441 Zoosyst. Evol. 100 (2) 2024, 437-445 ‘QOUDIOJOI WOI 9TQeIIeAR JOU “WN 1c0¢ ‘IP J9 4NANS| “(8 T6T) Iesngey pue Wo, 8961 slol 8E6l Oley Pr6T Oey) -(2G8T) uosdwns (78ST) sue] Apnjs siyj) — snaJe jj pue snove|| EG6T UeWAH 6G6T UeWAH| Iyeunqey pue aA} 9dUEJ2JoY ueder ‘ewiysno Wey ‘esnyewiy “EWRYEIIUS ueder ‘esnyewiy ‘EDOWIUS YSN ‘epuo}4 ueder ‘esnyewiy ‘1yeZ-a0w0| |ueder ‘OJON ‘IYeZON ‘IYeSI ‘OJON Ajey ‘sajden PUIUD ‘SUOPINH] ‘SpueJaUjay ‘auleuog YSN ‘elusosyen}! 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Prosthiostomum huidongense sp. nov., MBM287880 (holotype), schematic diagram (A) and photomicrographs of sagittal sections (B—E), anterior to the left. A. Copulatory complex and sucker; B. Anterior end of the body; C. Penis stylet; D. Male copu- latory apparatus; E. Copulatory complex and sucker. Abbreviations: ab — anterior branch of main intestine; br — brain; eg — cement glands; ep—cement pouch; fg — female gonopore; it — intestine; m — mouths; ma — male atrium; mg — male gonopore; ph — pharynx; pv — prostatic vesicle; spv — spermiducal vesicle; st — stylet; su — sucker; sv — seminal vesicle; va — vagina; ut — uterus. Scale bars: 0.5 mm (A, E); 200 um (B); 50 um (C, D). The new species has few eyes scattered between the marginal band and the cerebral eyes and thus can be easily distinguished from most congeners. Only five known species have been described with the above characteristics: P. awaensa Yeri & Kaburaki, 1918, P. griseum Hyman, 1959, P. latocelis Hyman, 1953, P. milcum Marcus & Marcus, 1968, and P. notoensis Kato, 1944. However, these species can be easily dis- tinguished from the new species by the dorsal color- ation or pigmentation pattern (except P. milcum) and the number of the marginal eyes (see Table 1). In ad- dition, P. awaensa, P. griseum, and P. latocelis are also different from P. huidongense sp. nov. in the position of the band of marginal eyes (distributed anterior to the brain for these species; extending to the level of the pos- terior margin of the brain in P. huidongense sp. nov.). zse.pensoft.net Although the dorsal coloration or pigmentation pattern of P. milcum is similar to that of the new species, it dif- fers from P. huidongense sp. nov. by the deep male atri- um and the distance between the female gonopore and sucker (Table 1). Prosthiostomum sonorum Kato, 1938, P. dohrnii Lang, 1884, P. nozakensis Kato, 1944, and P. grande also have yellow to brown mottles or spots on the dorsal surface, but they are different from P. huidon- gense sp. nov. in the arrangement of the eyes (Table 1). In general, identification of Prosthiostomum species is mainly achieved by features such as body size, dorsal color and pattern, eye arrangement and characteristics of the male and female reproductive system. Apart from that, the distance between the female gonopore and the sucker may be a noteworthy taxonomic feature with- in the genus, as in species P. latocelis, P. milcum and Zoosyst. Evol. 100 (2) 2024, 437-445 78/1 81/1 0.8 0.04 28S ML Prosthiostomum lobatum Euprosthiostomum mortenseni 443 400/1 Prosthiostomum acroporae (USA) Prosthiostomum acroporae (Australia) Prosthiostomum cf. ostreae 4100/1 Prosthiostomum trilineatum Prosthiostomum torquatum Prosthiostomum purum 88) 84/1 Prosthiostomum vulgare 76/0.94 Prosthiostomum auratum Prosthiostomum hibana 98 94/1 Prosthiostomum utarum Prosthiostomum cynarium Prosthiostomum grande Prosthiostomum milcum Prosthiostomum siphunculus Prosthiostomum sp. Prosthiostomum huidongense sp. nov. Prosthiostomum Euprosthiostomum Enchiridium periommatum 0.95}! Enchiridium evelinae u 100/1 83/0.96 | 0.85] Enchiridium sp.1 Enchiridium magec Enchiridium daidai 0.92 Enchiridium sp. 2 85/1 Enchiridium sp. 3 Enchiridium japonicum Enchiridium Enchiridium sp. 4 Pseudobiceros stellae Outgroup Figure 4. Maximum likelihood phylogenetic tree of species of the family Prosthiostomidae based on 28S rDNA. For clades that received either >70% ML bootstrap or >0.80 BI posterior probability, the support values are presented at the nodes. P. sonorum this distance differs from that in the new species (see Table 1). Our molecular phylogenetic analysis of Prosthiosto- midae based on partial 28S rDNA is largely consistent with that of Tsuyuki et al. (2021), in which the genera Prosthiostomum and Enchiridium form well-supported taxa. Although some Enchiridium species (e.g., E. ev- elinae, E. japonicum and E. punctatum) have similar dorsal patterns to that of P. huidongense sp. nov., they can be easily distinguished from the new species by lacking a muscular sheath that completely encloses the prostatic vesicles. This feature distinguishing these two genera is further corroborated by the molecular phylo- genetic tree. Euprosthiostomum mortenseni Marcus, 1948 is sister to the Prosthiostomum clade, with poor support (Fig. 4). According to Faubel (1984), absence of the frontal branch of the main intestine in Eupros- thiostomum distinguishes this genus morphologically from Prosthiostomum, but whether that character can be reliably used to discriminate the genus has not been clarified in the present study due to the limited number of available sequences (only one sequence available for Euprosthiostomum). Prosthiostomum huidongense sp. nov. 1s nested in the clade Prosthiostomum, thus corroborates our generic assignment based on the morphology. Uncorrected p-dis- tance of COI among 11 species/molecular entities of Pros- thiostomum are 5.7—24.3% (Suppl. material 1: table S2), while the average interspecific distance is 19% among these Prosthiostomum species that are morphologically distinguishable from each other. The uncorrected p-dis- tance between P. huidongense sp. nov. and other tested species ranged from 20.3 to 24.3%, which is greater than the average interspecific distance among Prosthiostomum species, thus, provided further support for the notion that P. huidongense is a new species. Acknowledgements This study was supported by grants from the Scientific and Technical Innovation Council of Shenzhen Gov- ernment (grant nos. jcyj20210324093412035 and kcex- £z20201221173404012), Innovation Team Project of Universities in Guangdong Province (No. 2023KCX- TD028) and China Undergraduate Training Program for Innovation and Entrepreneurship (S202310590068). We sincerely thank Prof. Ronald Sluys (Naturalis Biodiver- sity Center, P.O. Box 9517, 2300 RA Leiden, The Neth- erlands) for providing valuable comments on this work. zse.pensoft.net 4A4 References Barton JA, Bourne DG, Humphrey C, Hutson KS (2020) Parasites and coral-associated invertebrates that impact coral health. 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Invertebrate Biology 139(1): e12282. https://doi.org/10.1111/ ivb. 12282 4A5 Yeri M, Kaburaki T (1918) Description of some Japanese polyclad Tur- bellaria. Journal of the College of Science, Imperial University of Tokyo 39 Art. 9: 1-54. Supplementary material | Supplementary information Authors: Hai-Long Liu, Da-Hao Lin, An-Tai Wang, Zhang-Li Hu, Yu Zhang Data type: docx Explanation note: table S1. GenBank accession numbers of sequences for species taxa used in the phylogenetic analyses or genetic distance calculation; table S2. In- terspecific uncorrected p-distances for the COI gene fragments; figure S1. The tree is reconstructed by Bayesian inference analyses based on the 28S rDNA. 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.100.114482.suppl1 zse.pensoft.net