Zoosyst. Evol. 99 (2) 2023, 519-533 | DOI! 10.3897/zse.99.109097 > PENSUFT. yee BERLIN A new species of free-living marine nematode, Fotolaimus cavus sp. nov. (Nematoda, Oncholaimida, Oncholaimidae), isolated from a submarine anchialine cave in the Ryukyu Islands, southwestern Japan Daisuke Shimada! *, Keiichi Kakui!, Yoshihisa Fujita? 1 Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan 2 Center for Molecular Biodiversity Research, National Museum of Nature and Science, Tsukuba 305-0005, Japan 3 Okinawa Prefectural University of Arts, 1-4 Shuri-Tounokura, Naha, Okinawa 903-8602, Japan https://zoobank. org/7558 9A 9A-94C3-4F 20-BBEC-A4C7D3799836 Corresponding author: Daisuke Shimada (oncholaimus@gmail.com) Academic editor: Pavel Stoev Received 6 July 2023 # Accepted 6 October 2023 Published 6 November 2023 Abstract Fotolaimus cavus sp. nov. was described from a submarine anchialine cave called Akuma-no-yakata on the Shimoji Island, Miyako Island Group, Ryukyu Islands, southwestern Japan. This is the first free-living marine nematode isolated from a submarine cave in Japan, and the third species of the genus Fotolaimus. This new species differs from its congeners by its small body size, wide amphids, long buccal cavity, long conico-cylindrical tail, and proximally curved gubernaculum. We provide amended dichotomous keys to genera in the subfamily Oncholaiminae and species in Fotolaimus. We also analyzed partial DNA sequences encoding ribo- somal small subunit RNA and cytochrome c oxidase subunit I from Fotolaimus cavus sp. nov. and six other species of Oncholaim- idae collected from Japanese waters. The phylogenetic tree based on the ribosomal small subunit RNA sequences using maximum likelihood analysis suggested a close relationship between Fotolaimus and Wiesoncholaimus as well as Oncholaimus. The topology of the tree was similar to those from previous studies; however, it suggested a new phylogenetic position of Adoncholaimus as a sister clade for Viscosia and Oncholaimus. Key Words cave scuba diving, Enoplea, meiofauna, Miyako Island Group, molecular phylogeny, Oncholaiminae Introduction Free-living nematodes are the most abundant taxon in the marine environment and the dominant organisms in the meiobenthos of submarine caves, including anchi- aline caves (D’Addabbo et al. 2008; Arunimaand and Mohan 2021). However, surveys of meiofauna, includ- ing nematodes, in submarine caves have only been con- ducted in the Canary Islands (Garcia-Valdecasas Huelin 1985; Riera et al. 2018), Hong Kong (Zhou and Zhang 2003, 2008), Italy (Wieser 1954; Ape et al. 2015; On- orato and Belmonte 2017), and Cuba (Pérez-Garcia et al. 2018). Thus, our taxonomic and ecological knowl- edge of nematodes from submarine caves is insufficient. In the present study, we describe a new species of the free-living marine nematode genus Fotolaimus Bel- ogurova & Belogurov, 1974 collected from an anchial- ine cave called Akuma-no-yakata (= Devil’s Palace) and located on the Shimoji Island (or Shimojijima Island), Miyako Island Group, Ryukyu Islands, southwestern Ja- pan. Osawa and Fujita (2019) have provided a detailed description of this cave, and faunal surveys conducted in Akuma-no-yakata in recent years identified the fol- lowing organisms in the cave: crustaceans (Fujita et al. 2013, 2017; Anker and Fujita 2014; Osawa and Fujita 2016, 2019; Kakui and Fujita 2018, 2020; Saito and Fu- jita 2022), ophiuroids (Okanishi and Fujita 2018, 2019), annelids (Worsaae et al. 2021), molluscs (Mizuyama et Copyright Daisuke Shimada 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. 520 al. 2022), sponges (Ise et al. 2023), and pycnogonids (Kakui and Fujita 2023). Belogurova and Belogurov (1974) established the genus Fotolaimus in Oncholaimidae Filipjev, 1916. Currently, this genus contains two species, 1.e., F) mari- nus Belogurova & Belogurov, 1974 (type species) and F. apostematus (Wieser, 1959) Belogurova & Belogurov, 1974. Fotolaimus belongs to the subfamily Oncholaimi- nae Filtpjev, 1916, based on its left ventrosublateral tooth being larger than the other two teeth and the presence of a prodelphic ovary. Members of Foto/aimus resemble those of Metoncholaimus Filipjev, 1918 as males have a guber- naculum and females have a Demanian system, but differ from the Metoncholaimus species by the presence of ten or more terminal ducts and pores (whereas Metoncholaimus members have two ducts and two pores) (Belogurova and Belogurov 1974; Belogurov and Belogurova 1988; Smol et al. 2014). However, there are currently no molecular data supporting the phylogenetic position of Fotolaimus. In a recent classification system by Hodda (2022), the family Oncholaimidae belongs to the order Oncholaimi- da Siddiqi, 1983 = suborder Oncholaimina De Coninck, 1965 = superfamily Oncholaimoidea Filipjev, 1916 with two other families, i.e., Enchelidiidae Filipjev, 1918 and Thalassogeneridae Orton Williams & Jairaypuri, 1984. Thalassogeneridae is a terrestrial family that includes only the type genus 7halassogenus Andrassy, 1973, and several authors do not include Thalassogeneridae in On- cholaimoidea based on morphological data (Jensen 1976; Lorenzen 1981). Since molecular data do not allow any conclusion, only Oncholaimidae and Enchelidiidae are considered members of Oncholaimoidea sensu stricto. Several molecular phylogenetic analyses strongly sup- port the monophyly of the clade composed of Oncholai- moidea sensu stricto (Meldal et al. 2007; van Megen et al. 2009; Bik et al. 2010a, b; Pereira et al. 2010; Smythe 2015; Smythe et al. 2019; Ahmed et al. 2022). However, analyses of ribosomal small subunit RNA sequences (Meldal et al. 2007; Bik et al. 2010a, b; Smythe 2015) and whole-genome/transcriptome (Smythe et al. 2019; Ahmed et al. 2022) do not support monophyly of On- cholaimidae. The monophyly of the seven subfamilies comprising Oncholaimidae (for three of which there are no molecular data) is also not supported by molecular analyses (Bik et al. 2010a, b; Smythe 2015), and phy- logenetic relationships within Oncholaimoidea that have been supported by morphological analyses (e.g., Smol et al. 2014; Hodda 2022) are considered to require signifi- cant revision. Materials and methods Sampling and morphological observation Sediment samples were collected by scuba diving from a completely dark, anchialine zone at 7-20 m depth (“second slope zone,” Osawa and Fujita 2019) in the zse.pensoft.net Daisuke Shimada et al.: A new submarine cave nematode from Japan submarine cave Akuma-no-yakata, Shimoji Island, Mi- yako Island Group, Ryukyu Islands, southwestern Japan (24°49'22.5"N, 125°08'07.8"E) on 26 Oct. 2018. Thirteen nematode individuals were isolated from the sediments and preserved in DESS solution (Yoder et al. 2006). We considered that these individuals belonged to the same species in the family Oncholaimidae. Three males and four females were permanently mounted in anhydrous glycerin (Shimada et al. 2021), observed using a BX51 light microscope (Olympus, Japan) with differential in- terference contrast, and photographed with an PCM500 digital camera (AS ONE, Japan). One male and one fe- male were dried using the hexamethyldisilazane method (Nation 1983), sputter-coated with gold to a thickness of 20 nm, and observed using an S-3000N scanning elec- tron microscope (SEM; Hitachi, Japan). We edited digital photographs using GIMP ver. 2.10 (https://www.gimp. org) and generated measurements and drawings from digital photographs using Inkscape ver. 1.0 (https://ink- scape.org). We deposited all specimens examined in the Invertebrate Collection of Hokkaido University Museum (ICHUM). The terminology used to describe the arrange- ment of morphological features such as setae follows that of Decraemer et al. (2014). The following de Man’s ratios (Hooper 1986) were used: a, ratio of body length to maxi- mum body diameter; b, ratio of body length to pharyngeal length; c, ratio of body length to tail length; c’, ratio of tail length to body diameter at cloacal opening or anus; and V, position of vulva from anterior body end expressed as percentage of body length. Molecular experiments Two males and two females isolated from the Aku- ma-no-yakata cave were used. Additionally, we includ- ed 18 individuals of six oncholaimid species from Jap- anese waters, 1.e., Oncholaimus secundicollis Shimada, Kajithara, & Mawatari, 2009, Oncholaimus cf. oxyuris Ditlevsen, 1911, Oncholaimus cf. vesicarius (Wieser, 1959), Wiesoncholaimus jambio Shimada & Kaku, 2021, Adoncholaimus daikokuensis Shimada & Kayjihara, 2014, and Adoncholaimus pseudofervidus Shimada & Kajiha- ra, 2014, for phylogenetic analysis (Table 1). Total DNA was extracted using an ISOHAIR extraction kit (NIPPON GENE, Japan) following the protocols described by Tana- ka et al. (2012) and Iwahori (2014) with minor modifica- tions. Each nematode was placed in 20 uL dissolving solu- tion containing 18.5 wL 1% extraction buffer in TE buffer pH 8.0, 0.5 uL lysis solution, and 1.0 wL enzyme solution and incubated at 55 °C for 60 min (extraction buffer, ly- sis solution, and enzyme solution are included in the kit). Nearly full-length sequences encoding ribosomal small subunit RNA (18S) and mitochondrial cytochrome c oxi- dase subunit I (COI) were amplified by PCR using KOD One PCR Master Mix (TOYOBO, Japan). PCR primers for 18S amplification were as follows: forward EukA (AACCTGGTTGATCCTGCCAGT) (Diez et al. 2001) Zoosyst. Evol. 99 (2) 2023, 519-533 opal Table 1. List of nematodes sequenced from Japanese waters with INSD accession numbers. *Type locality. Species N Date Fotolaimus cavus sp. nov. 4 26 Oct. 2018 Wiesoncholaimus jambio 4 25 Jun. 2015 Oncholaimus secundicollis 2 13 Jul. 2014 Oncholaimus cf. oxyuris 5 23 Jun. 2013 Oncholaimus cf. vesicarius 18 17 Mar. 2015 Adoncholaimus daikokuensis 4 19 Jun. 2012 Adoncholaimus pseudofervidus 4 20 May 2012 and reverse 26R (CATTCTTGGCAAATGCTTTCG), forward 9FX (AAGTCTGGTGCCAGCAGCCGC) and reverse 13R (GGGCATCACAGACCTGTTA), and for- ward 2FX (GGAAGGGCACCACCAGGAGTGG) and reverse 18P (TGATCCWKCYGCAGGTTCAC) (De Ley et al. 2002). PCR primers for COI amplification were forward Fl (CCTACTATGATTGGTGGTTTTGGTA- ATTG) and reverse R2 (GFAGCAGCAGTAAAATA- AGCACG) (Kanzaki and Futai 2002) and forward JB3 (TTTTTTGGGCATCCTGAGGTTTAT) and _ reverse JB5 (AGCACCTAAACTTAAAACATAATGAAAATG) (Derycke et al. 2005). The thermal cycling program con- sisted of 35 cycles at 98 °C for 10 s, 50 °C for 5 s, and 68 °C for 10 s. We determined the nucleotide sequenc- es by direct sequencing using a BigDye Terminator Kit ver.3.1 (Applied Biosystems, USA) with a 3730 Genetic Analyzer (Applied Biosystems). Fragments were joined into a single sequence using MEGA X (Kumar et al. 2018). We deposited the obtained sequences (Table 1) in the International Nucleotide Sequence Database (INSD) through the DNA Data Bank of Japan. Phylogenetic analysis The 18S dataset (1442 bp long after alignment) used for phylogenetic analysis included 21 oncholaimid sequenc- es obtained in the present study, and 38 oncholaimid se- quences, 12 enchelidiid sequences, and three outgroup sequences (Enoplus Dujardin, 1845 and Enoploides Ssaweljev, 1912) obtained from the INSD (Table 2). All sequences were aligned according to the secondary struc- ture predicted using RNAfold WebServer (Gruber et al. 2008; Lorenz et al. 2011). Sequences were trimmed in MEGA X and alignment-ambiguous sites were then re- moved using Gblocks ver. 0.91b (Castresana 2000) in NGPhylogeny.fr (Lemoine et al. 2019) and the “relaxed” parameters described in Talavera and Castresana (2007). The optimal substitution model was GTR+F+R3, deter- mined under the corrected Akaike information criterion option in ModelFinder (Kalyaanamoorthy et al. 2017). A maximum likelihood (ML) analysis was construct- ed based on the 18S dataset using IQ-TREE ver. 2.1.2 (Minh et al. 2020) under the “bnni’ option (Hoang et al. 2018). Clade support was estimated using 1,000 repli- cates for both SH-like approximate likelihood ratio tests (SH-aLRT; Guindon et al. 2010) and ultrafast bootstraps Locality Akuma-no-yakata “off Misaki, Sagami Bay *Akkeshi, Hokkaido Hamanaka, Hokkaido Akkeshi, Hokkaido *Daikoku Island, Hokkaido *Mukawa, Hokkaido Accession numbers 18S Col LC746839-LC746842 LC746861-LC746864 LC746843-LC746846 LC759639-LC759641 LC746847, LC 746848 - LC746849, LC746850 LC746865-LC 746869 LC746851, LC746852 LC746870-LC 746887 LC746853-LC746856 LC746888-LC746891 LC746857-LC746860 LC746892-LC746895 (UFBoot; Hoang et al. 2018) as a function of IQ- TREE. The ML tree was drawn using FigTree ver. 1.4.4 (http:// tree.bio.ed.ac.uk) and processed using Illustrator CS6 (Adobe, USA). Results Taxonomy Subfamily Oncholaiminae Filipjev, 1916 Type genus. Oncholaimus Dujardin, 1945. Diagnosis (modified from Belogurova and Bel- ogurov 1974; Smol et al. 2014). Oncholaimidae. Cuticle smooth. Buccal cavity barrel-shaped, with three teeth. Left ventrosublateral tooth larger than other teeth (usu- al) or left and right ventrosublateral teeth of same size. Spicules short or long, gubernaculum present or absent. Copulatory bursa absent. Female reproductive system monodelphic-prodelphic with an antidromously reflexed ovary. Demanian system present or absent. Remarks. Cobb (1930) established the new genus On- cholaimium Cobb, 1930, which differs from Oncholaimus mainly by the presence of a distinct precloacal appendi- cule (papilla) in males. Subsequently, Kreis (1932) estab- lished Pseudoncholaimus Kreis, 1932 based on the lack of Demanian system. Kreis (1934) also distinguished On- cholaimium from Oncholaimus based on the Demanian system, which is without terminal pore in Oncholaimi- um and with terminal pores in Oncholaimus. However, Rachor (1969) synonymized Oncholaimium and Pseud- oncholaimus to Oncholaimus, because the presence of a Demanian system is unknown for many species of Oncholaimus. Belogurov and Belogurova (1988), who proposed the currently accepted taxonomic system of Oncholaimidae, treated Oncholaimium and Pseudon- cholaimus as valid genera. Pseudoncholaimus 1s still considered valid by several researchers (e.g., Smol et al. 2014; Tsalolikhin 2015; Milovankina and Fadeeva 2019). However, because it is unclear for numerous species whether they rather belong to Oncholaimus, Oncholaim- ium, or Pseudoncholaimus, we included the latter two in Oncholaimus sensu lato as described by Rachor (1969). Thus, the subfamily Oncholaiminae comprises six gen- era, 1.e., Oncholaimus, Metoncholaimus, Prooncholaimus Micoletzky, 1924, Metaparoncholaimus De Coninck & zse.pensoft.net 522 Daisuke Shimada et al.: A new submarine cave nematode from Japan Table 2. List of nematode sequences from the INSD including the phylogenetic analysis. Abbreviations: A = Atlantic Ocean side; FS = French Southern and Antarctic Lands; nd = no data; P = Pacific Ocean side. *Outgroup. Family Subfamily Species Accession Reference number Oncholaimidae Oncholaiminae Oncholaimus sp. AUK23 UK HM564402 Bik et al. (2010b) Oncholaimus sp. AUK35 UK HM5644.74. Bik et al. (2010b) Oncholaimus sp. AUK36 UK HM564475 Bik et al. (2010b) Oncholaimus sp. BUS1 USA (A) HM564404 Bik et al. (2010b) Oncholaimus sp. BUS4 USA (A) HM564409 Bik et al. (2010b) Oncholaimus sp. BUS5 USA (A) HM564410 Bik et al. (2010b) Oncholaimus sp. BUS7 USA (A) HM564411 Bik et al. (2010b) Oncholaimus sp. NAR4 USA (A) HM564429 Bik et al. (2010b) Oncholaimus sp. NAR7 USA (A) HM564432 Bik et al. (2010b) Oncholaimus sp. NAR16 USA (A) HM564426 Bik et al. (2010b) Oncholaimus sp. NUS2 USA (A) HM564438 Bik et al. (2010b) Oncholaimus sp. NUS5 USA (A) HM564444 Bik et al. (2010b) Oncholaimus sp. NUS6 USA (A) HM564445 Bik et al. (2010b) Oncholaimus sp. NUS7 USA (A) HM564446 Bik et al. (2010b) Oncholaimus sp. OUS2 USA (A) HM564450 Bik et al. (2010b) Oncholaimus sp. SBA2 South Africa HM564592 Bik et al. (2010b) Oncholaimus sp. SBA3 South Africa HM564593 Bik et al. (2010b) Oncholaimus sp. SBA5 South Africa HM564594 Bik et al. (2010b) Oncholaimus sp. AS479 Japan KR265044. Smythe (2015) Oncholaimus sp. DS-2015 Japan LC093124 Shimada (unpubl.) Pseudoncholaimus sp. AS89 USA (A) KR265048 Smythe (2015) Adoncholaiminae Adoncholaimus sp. nd AF036642 Mullin et al. (2005) Oncholaimellinae Viscosia sp. AUK10 UK HM564399 Bik et al. (2010b) Viscosia sp. HCL9 UK HM564570 Bik et al. (2010b) Viscosia sp. HCL10 UK HM564557 Bik et al. (2010b) Viscosia sp. HCL11 UK HM564558 Bik et al. (2010b) Viscosia sp. HCL15 UK HM564560 Bik et al. (2010b) Viscosia sp. HCL24. UK HM564565 Bik et al. (2010b) Viscosia sp. HCL27 UK HM564566 Bik et al. (2010b) Viscosia sp. LUK1 UK HM564417 Bik et al. (2010b) Viscosia sp. LUK3 UK HM564419 Bik et al. (2010b) Viscosia sp. SBN2 UK HM564595 Bik et al. (2010b) Viscosia sp. SBN4 UK HM564597 Bik et al. (2010b) Viscosia dossena Leduc & Zhao, 2023 New Zealand OK317193 Leduc and Zhao (2023) Oncholaimellinae sp. AS71 USA (A) KR265043 Smythe (2015) Pontonematinae §Pontonema sp. Nem.209 USA (P) MN250102 ‘Pereira et al. (2020) Pontonema sp. Nem.213 USA (P) MN250105 +Pereira et al. (2020) Enchelidiidae Bathyeurystomina sp. Cr78a FS HM564537 Bik et al. (2010b) Bathyeurystomina sp. Cr80b FS HM564539 Bik et al. (2010b) Bathyeurystomina sp. TCR81 USA (P) HM564646 Bik et al. (2010b) Bathyeurystomina sp. TCR109 USA (P) HM564602 Bik et al. (2010b) Calyptronema sp. 1068 the Netherlands FJO40503 van Megen et al. (2009) Calyptronema sp. AUK13 UK HM564400 Bik et al. (2010b) Calyptronema sp. LUK7 UK HM564421 Bik et al. (2010b) Calyptronema sp. LUK12 UK HM564418 Bik et al. (2010b) Eurystomina sp. AS485 Japan KR265038 Smythe (2015) Pareurystomina sp. BCA3 Antarctica HM564491 Bik et al. (2010b) Pareurystomina sp. NUS1 USA (A) HM564435 Bik et al. (2010b) Symplocostoma sp. AS520 Panama (A) KR265050 Smythe (2015) Enoplidae *Enoplus brevis Bastian, 1865 nd U88336 Aleshin et al. (1998) *Enoplus meridionalis Steiner, 1921 Croatia Y16914 Kampfer et al. (1998) Thoracostomopsidae “Enoploides sp. 1252 the Netherlands FJO40490 van Megen et al. (2009) Schuurmans Stekhoven, 1933, Wiesoncholaimus Inglis, 1966, and Fotolaimus. Members of the Oncholaiminae can be distinguished from these of the other six subfam- ilies as follows. They are distinct from Krampiinae De zse.pensoft.net Coninck, 1965, Adoncholaiminae Gerlach & Riemann, 1974, and Pontonematinae Gerlach & Riemann, 1974 as they have only one ovary (whereas the three other gen- era have two); from Pelagonematinae De Coninck, 1965 Zoosyst. Evol. 99 (2) 2023, 519-533 and Octonchinae De Coninck, 1965 as they have three distinct teeth (whereas Pelagonematinae member have minute or no tooth and Octonchinae have eight or more); and from Oncholaimellinae De Coninck, 1965 because their left ventrosublateral tooth is larger than the other two teeth (right ventrosublateral tooth is larger in On- cholaimellinae). Recent revisional works were provided by Mawson (1958) for Metaparoncholaimus, Yoshimura (1982) for Metoncholaimus, and Chen et al. (2015) for Prooncholaimus. Genus Fotolaimus Belogurova & Belogurov, 1974 Type species. Fotolaimus marinus Belogurova & Bel- ogurov, 1974. Diagnosis (modified from Belogurova and Belogurov 1974). Oncholaiminae. Left ventrosublateral tooth larger than the two other teeth. Spicules shorter than 2.0 cloacal body diameters. Gubernaculum present. Demanian system present, posterior end of main duct forming two symmetri- cal sacs each with five or more terminal ducts. Remarks. The genus Foto/aimus can be distinguished from Metaparoncholaimus and Wiesoncholaimus by the left ventrosublateral tooth being larger than the right ventrosublateral tooth (left and right ventrosublater- al teeth are equal in size in Metaparoncholaimus and 523 Wiesoncholaimus). It also differs from Prooncholaimus by the absence of the large bubble-like cells in pseudo- coelom (presence in Prooncholaimus). Fotolaimus, On- cholaimus, and Metoncholaimus are similar to each other and are distinguished by the morphological characters of the terminal ducts of the Demanian system. Belogurova and Belogurov (1974) characterized the Demanian sys- tem of Fotolaimus as follows: the posterior end of the main duct forms two symmetrical sacs, each of which is pierced by five terminal ducts ending in terminal pores. On the other hand, the main ducts of Oncholaimus and Metoncholaimus do not form a sac at the posterior end, but branch separately into two or more terminal ducts (Belogurov and Belogurova 1988). Belogurov and Bel- ogurova (1977) distinguished Metoncholaimus from Oncholaimus in that the terminal ducts are covered with moniliform glands. However, some Metoncholaimus spe- cies do not mention the shape of the terminal duct in their description (e.g., Mawson 1958; Salma et al. 2017). For convenience, a species may be considered belonging to Oncholaimus if it has short (equal to cloacal body diam- eter) spicules and without gubernaculum, and belonging to Metoncholaimus if it has longer spicules (with or with- out gubernaculum) or short spicules with gubernaculum (cf. Platt and Warwick 1983). The distinction between Oncholaimus and Metoncholaimus will need to be reex- amined in the future. Key to genera in Oncholaiminae (cf. Belogurov and Belogurova 1988; Smol et al. 2014). 1 Rightranchiett-ventrosubblateralsteethpor -SamesSiZer 223.4... an Wade ctgcacesshadabasds Mcccdes oenen des ads undecdacqeestutl deseds Meidessabexed 2 - Eett-Ventresublarcral toon larserathanictie-Omiernkeew |, cu) a tah Iisa Ale clas oa, cae al a oa ed ones Ade Oe ls ares 3 2 Spicute-lenetiival most-equalatocc|oacal body clamelek 2072: texto 2s, ae ee ee pas Metaparoncholaimus - Spiculessongerthar o-0-clodcali Poay Cialmetens. ra sete rth eee ness Paley ee oa hres Sees Pe Pre ee Seer Wiesoncholaimus 3 larse bUBDbIC- like CellSipresent: IM PSSUGOCOSI OI o.5.5. .bcmeancpenntortinides sstdhamlcetenans samdeds shane agteersces gy names sceneries Prooncholaimus - ker oe DUDBISIKe:Celll Sab Semin Foe i, Ok i Ee ar ence ey aneten en eae atethe Se sok: BE 4 4 DermMaiiianinsySteniihel WGiiialWOoPOSTOHIOMSaCSs.5 sc. ict. fy PRL ccc h onde cso ewe eects yy Pa se eee Fotolaimus ~ Pemanian System -absenit, OM presents belle witOUt: POSTENOP SACS ae. 6.6. fie cng campeiese hep aneldan dines ngvvntiow pant hd doce agseapebonbug ene dake 5 bus FVerimindl. auets Cove roc ew itiiinmO muon Plan Ss25.4--+.tsnneeenee nae hgdedyaakhcurtdaak Seedbdancdns rasurty tetas sig@ha dere, axknemtie Metoncholaimus - Terminal ducts not covered with moniliform glands Fotolaimus cavus sp. nov. https://zoobank.org/A0509360-77DD-4387-9FA D-92B14758952F Figs 1-4, Table 3 Material examined. Holotype. JAPAN * male (perma- nent whole mount in glycerin); Ryukyu Islands, Miyako Island Group, Shimoji Island, a submarine cave called Akuma-no-yakata; 24°49'22 5"N, 125°08'07.8"E; 26 Oct. 2018; anchialine zone, depth 7 m, collected by Yoshihisa Fujita; ICHUM 8474. Paratypes. JAPAN * two males (permanent whole mounts in glycerin); same collection data as for holotype; 26 Oct. 2018; anchialine zone, depth 20 m, collected by Yoshihisa Fujita; ICHUM 8475 and 8476 « four females (permanent whole mounts in glycerin); same collection data as for preceding; ICHUM 8477-8480. tite, SS Ne RISES ed Pe Pn etree ee wae Se PWS SPE RTE Pe: Oncholaimus Other material. JAPAN * one male (SEM specimen); same collection data as for paratypes; * one female (SEM specimen); same collection data as for preceding. Etymology. The specific name cavus (cave) is a Latin noun in apposition derived from the type locality. Description. Body (Fig. 1A, B) elongated, almost cy- lindrical but gradually tapering toward both extremities. Cuticle smooth throughout body besides oblique stria- tions (Fig. 2A; cf. Leduc 2013) crossing at angle of ca. 120° between amphids and anteriormost cervical setae visualized using SEM. Somatic sensilla arranged in eight longitudinal rows: setiform in anterior half of cervical (Figs 1C, 2B) and caudal (Fig. 2C) regions; papilliform or very short setiform in rest of body (Fig. 2D), difficult to observe without SEM. Cephalic region (Figs 1D—F, 2E— G, 3A—D) rounded at anterior end, with six lips, slightly zse.pensoft.net 524 Daisuke Shimada et al.: A new submarine cave nematode from Japan C ges Ne E ve —— CC : CEA Ary leqeen t{| 7 — Nh F > 6 / Figure 1. Line drawings of Fotolaimus cavus sp. nov. A, C, D. Holotype (CHUM 8474); B, E. Paratype (ICHUM 8477); F, Para- type (CHUM 8479). A. Male body, right lateral view; B. Female body, right lateral view; C. Male cervical region, right lateral view; D. Male cephalic region, right lateral view; E. Female cephalic region, right lateral view; F. Female cephalic region, dorsal view. Scale bars: 1 mm (A, B); 100 um (C); 20 um (D-F). zse.pensoft.net Zoosyst. Evol. 99 (2) 2023, 519-533 525 Figure 2. SEM photographs of Fotolaimus cavus sp. nov. A, B, D-F, M—O. Non-type female; C, G—L. Non-type male. A. Oblique striations on cuticle; B. Anterior region; C. Male cloacal region; D. Papilliform somatic sensilla (arrowheads); E. Female cephalic region, lateral view; F. Female cephalic region, anterior view; G. Male cephalic region, anterior view; H. SE-pore; I. Male posterior region with cloacal opening (arrowhead); J. Tail tip with spinneret (arrowhead); K. Cloacal region, subventral view; L. Ventral papillae; M. Vulva; N. Circle of terminal pores; O. Terminal pores. Scale bars: 5 um (A, H, J, L); 50 um (B, DI; 20 um (C, E, N); 10 um (D, F, G, K, M, O). zse.pensoft.net 526 constricted posterior to amphids, as wide as 0.3—0.4 max- imum body diameters at cephalic sensilla level. Six inner labial sensilla papilliform. Six outer labial and four ce- phalic sensilla setiform, arranged in single circle, 5-8 um or 0.20—0.30 corresponding body diameters long in males and 6—9 um or 0.25—0.35 corresponding body diameters long in females. Amphids (Figs 1D, E, 2E, 3B) pocket-like, with elliptical aperture and cup-shaped fovea, 0.40—0.45 corresponding body diameters wide in males and 0.35— 0.40 corresponding body diameters wide in females, zse.pensoft.net Daisuke Shimada et al.: A new submarine cave nematode from Japan Figure 3. Light micrographs of Foto/aimus cavus sp. nov. A-F, H, J. Holotype (CHUM 8474); G, K. Paratype (ICHUM 8477); I. Paratype (CHUM 8475); L. Paratype (ICHUM 8479). A. Anterior region; B. Amphid; C. Buccal cavity with left ventrosublat- eral tooth; D. Buccal cavity with right ventrosublateral tooth; E. Posterior end of pharynx with cardia; F. Male tail; G. Female tail; H, I. Spicule (black arrowhead) and gubernaculum (white arrowhead); J. Sperms in seminal vesicle; K. Uvette and ductus entericus; L. Terminal pores. Scale bars: 20 um (A, E, H—-K); 10 um (B—D, L); 100 um (F, G). anterior margin located at 0.4—0.5 buccal cavity lengths from anterior body end. Buccal cavity (Figs 1D—F, 3C, D) barrel-shaped, length/width = 2.5—3.0, surrounded by pharyngeal tissue in posterior 15%-—25%. Three well-de- veloped teeth: left ventrosublateral tooth largest, 4-6 um longer than right and dorsal teeth. Tip of left ventrosublat- eral tooth at 0.2 buccal cavity lengths from anterior body end (1.e., level of cephalic sensilla). Pharynx (Figs 1C, 3E) cylindrical, evenly muscular, gradually widened toward posterior end. Cardia surrounded by intestine. Rectum Zoosyst. Evol. 99 (2) 2023, 519-533 527 A SS i} —|_4 A pn » 90% and UFBoot = 100%); (3) Calyptronema Marion, 1870 and Symplocostoma Bastian, 1865 (SH-aLRT = 94.8% and UFBoot = 97%); and (4) parts of Oncholaimus with all Wiesoncholaimus, Fotolaimus, Meyersia Hopper, 1967 and Oncholaimellinae sp. (SH-aLRT = 90.7% and UFBoot = 90%). The support values for a second major clade were not well-supported (SH-aLRT < 70% and UFBoot < 80%). Discussion The 18S phylogenetic tree (Fig. 5) suggested that Foto- laimus cavus sp. nov. was assembled in one major clade with some members of Oncholaimus and all members of Basia ROU ey OE OF aR acy se ner ee F, apostematus pits do Perret ele sco ter aie, 2 eae eer F, Cavus Sp. nov. Wiesoncholaimus and Oncholaimellinae sp. with a high level of support (SH-aLRT = 96.3% and UFBoot = 97%). Fotolaimus and Wiesoncholaimus are included, with Oncholaimus, in the subfamily Oncholaiminae (Hodda 2022), and they all present an oncholaimid-type Demanian system (Belogurov and Belogurova 1988). Therefore, the position of Fotolaimus in the Oncholaiminae based on the morphology was supported by molecular phylogeny. Oncholaimus sp. (KR265044) from Wakayama (Pacif- ic side of central Japan) is considered to be O. secundicol- lis because DNA sequences obtained from the former was identical to those from the topotypes of O. secundicollis. Oncholaimus secundicollis is distributed on the Pacific and Sea of Japan sides of northeastern Japan (Shimada unpubl.) and on the Sea of Japan side of South Korea (Lee et al. 2015). Wieser (1955) also reported on O. dujardinii de Man, 1876 from Wakayama but provided limited mor- phological information and no illustration. A redescrip- tion of O. dujardinii by the same author (Wieser 1953) mentions a gubernaculum (Zhang and Platt 1983), which should be absent in Oncholaimus. Therefore, O. dujar- dinii sensu Wieser (1955) may not be a true Oncholaimus. Meyersia branch has been assembled (SH-aLRT = 90.7% and UFBoot = 90%) with the Oncholaimus—Wi- esoncholaimus—Fotolaimus clade. Meyersia did not form the clade with Adoncholaimus, indicating that monophyly of Adoncholaiminae is unlikely. Members of Adoncholaiminae possess two ovaries and a well-de- veloped Demanian system, but other morphological fea- tures distinguish Meyersia from the other three genera. Adoncholaimus (including Metoncholaimoides Wieser, 1954), Admirandus Belogurov & Belogurova, 1979, and Kreisoncholaimus Rachor, 1969 have larger teeth on the right side, and the terminal pores of the Demanian system are located near the anus (1.e., much posterior to both ova- ries). In contrast, in Meyersia, right and left teeth are large, and the terminal pores of the Demanian system are located at the level of the vulva (i.e., between both ovaries). Monophyly of Enchelidiidae was not supported by our phylogenetic tree, although previous studies by Bik et al. (2010a, b) evidenced monophyly with 95% of bootstrap zse.pensoft.net 530 95.9/95 Daisuke Shimada et al.: A new submarine cave nematode from Japan Q| Adoncholaimus daikokuensis Adoncholaimus sp. AF036642.1 78.9/91 © Adoncholaimus pseudofervidus Oncholaimus sp. (OUS) — Viscosia dossena 95.9/95 I © Viscosia sp. (SBN) = Bide Viscosia sp. (AUK, HCL, LUK) Pseudoncholaimus sp. KR265048.1 69? Oncholaimus sp. (NAR) Q Oncholaimus sp. (BUS, NUS, SBA) Pontonema sp. Eurystomina sp. KR265038.1 | 4 Pareurystomina sp. HMS64435.1 Enchelidiidae Pea 95 4/99 Pareurystomina sp. HM564491.1 Adoncholaiminae 95/85 ; Bathyeurystomina sp. HM564539. 1 Oncholaimellinae ? Bathyeurystomina sp. HM564537.1 Oncholaiminae 1 A ea Bathyeurystomina sp. HM564602.1 Pontonematinae aa Bathyeurystomina sp. HM564646.1 Sooo 89.2/91 Calyptronema sp. FJ040503.1 “9A 8/97 Calyptronema sp. (AUK, LUK) Symplocostoma sp. KR265050. 1 Meyersia sp. KR265042.1 LC746839 LC746841 : 90.7/90 LC746842 Fotolaimus cavus sp. nov. LC746840 96. 3/97 Oncholaimus cf. vesicarius © Wiesoncholaimus jambio Oncholaimus secundicolles 2014AK04 SH-aLRT = 100; 4 Oncholaimus sp. KR265044.1 UFBoot = 100 Oncholaimus secundicolles 2014AK05 9 100 > SH-aLRT = 90; UFBoot = 100 95.7/80 0.04 Oncholaimus sp. (AUK) 9 Oncholaimus cf. oxyuris Oncholaimus sp. LC093124.1 Oncholaimellinae sp. KR265043.1 Figure 5. ML tree of Oncholaimoidea based on 18S DNA sequences. Numbers at the nodes are SH-aLRT (left) greater than 70% and UFBoot (right) greater than 80%. values. Monophyly of the clade comprising Calyptrone- ma and Symplocostoma was well supported (SH-aLRT = 94.8% and UFBoot = 97%). Both genera present a sexually dimorphic cephalic region, elongated spicules, and papilli- form (not winged) precloacal supplements, and have been considered, together with Symplocostomella Micoletzky, 1930, which has the same three characteristics, closely re- lated groups within Enchelidiidae. Pontonema appeared to be a sister group of the clade consisting of Eurystomina and Pareurystomina Micoletzky, 1930; however, the support values are quite low (SH-aLRT < 70% and UFBoot < 80%), consequently, the position of Pontonema remains uncertain. Some members of Oncholaimus and all members of Pseudoncholaimus and Viscosia appear to be monophylet- ic (SH-aLRT = 97.3% and UFBoot = 99%). As aforemen- tioned, Pseudoncholaimus is considered a junior synonym of Oncholaimus, but both taxa can be distinguished by the presence or absence of Demanian system. Our tree strong- ly suggested that unidentified Oncholaimus spp. was not clustered in a single clade. The species of Oncholaimus zse.pensoft.net clustered with Pseudoncholaimus might not have a De- manian system. In fact, the species that doubtlessly had a Demanian system (O. secundicollis) belonged to a sep- arate clade from Pseudoncholaimus. Fotolaimus and Wiesoncholaimus, suggested to be closely related to O. secundicollis, also have a Demanian system. Therefore, there was no evidence supporting the synonymization of Pseudoncholaimus with Oncholaimus. Because members of Oncholaimus (in which the left tooth is the largest) are included in two well-supported clades, which both con- tain Oncholaimellinae spp. (in which the right tooth is the largest), it is likely that the size of the left and right teeth does not reflect phylogenetic relationships. Additionally, Wiesoncholaimus, in which left and right teeth are large, was in the same clade as Oncholaimus and Fotolaimus, suggesting that the teeth size has evolved independently many times. In Bik et al. (2010a, b) and Smythe (2015), the phyloge- netic position of Adoncholaimus within Oncholaimoidea was not clear. Our phylogenetic analysis suggested that Zoosyst. Evol. 99 (2) 2023, 519-533 Adoncholaimus 1s a sister clade of Oncholaimus—Pseud- oncholaimus—Viscosia clade with a certain degree of sup- port (SH-aLRT = 78.9% and UFBoot = 91%). Acknowledgements We wish to thank Hiroki Ichi (Irabu-jima Fishery Cooper- ative), Masaru Mizuyama (Meio University), Katrine Wor- saae (University of Copenhagen), Peter Rask Moller (Natu- ral History Museum Denmark, University of Copenhagen), and Go Tomitani (Diving Service “Marines Pro Miyako’) for helping with scuba diving and sampling in the submarine cave. 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