Description of a New Species of Eusynstyela (Ascidiacea: Stolidobranchia) from Okinawa, Japan with Molecular Insight into Its Phylogenetic Position Hasegawa, Naohiro Species Diversity 2025 2025-05-23 30 1 99 106 Hartmeyer, 1903 Hartmeyer 1903 [370,661,458,483] Ascidiacea Styelidae Animalia Stolidobranchia 3 102 Chordata subFamily Polyzoinae   Material examined.  Holotype: NCHSU 962, with one zooid removed for morphological observation and another for DNA extraction.  Paratypes: NCHSU 963–968, with one zooid from each paratype, also removed for morphological observation. The holotypeand one of the paratypes( NCHSU 963) were collected at Sesoko Islandby N. Hasegawa, NCHSU 964at Mizugama by Y.  Kushida of Rissho University, NCHSU 965–967at Mizugama by N. Hasegawa, and NCHSU 968at Maeda Cape by N. Hasegawa (Table 2).   Description.Colony sheet-shaped, attaching to coral rocks and other solitary ascidians ( Fig. 2A, B). Zooids 0.2– 1.3 cmin length, oval in outline ( Fig. 2C), basally connected to each other by ventral tunic. In life, tunic colored by yellow or light orange on dorsal side, orange or red on lateral side, two dark red lines connecting both siphons; tough and leathery, but thinner on ventral side ( Fig. 2A, B). Siphons four-lobed, reddish in life. After fixation in formalin, tunic color turned to brownish orange. Ten oral tentacles; almost uniformly sized, ca. 0.3 mmlong ( Fig. 2D). Neural gland positioned at base of oral siphon in dorsal body wall, opening into peripharyngeal cavity as longitudinal slit ( Fig. 2D). Dorsal lamina smoothly margined ( Fig. 2D). Four folds on each side of pharynx; first and third folds from dorsal lamina with two to three times more longitudinal vessels than second and fourth folds; exception in paratypeNCHSU 965: left-side folds with 14, 10, 10, and 6 vessels from dorsal to ventral, showing gradual decrease; right-side folds with 12 vessels on first fold, and 8, 9, 10 on second to fourth folds, minimal variation (Table 2). About ten stigmata per mesh between endostyle and first longitudinal vessel from endostyle. Intestine situated on left side, sharply curving from pyloric level, then again bending from cardiac part for running to atrial siphon ( Fig. 2C). Esophagus short. Globular stomach with 12 internal longitudinal folds occupying approximately half of intestinal loop; gastric caecum absent ( Fig. 2C). Spherical and hermaphroditic gonads located on ventral region; their number on left and right sides 7–15 and 7–20, respectively; all of them arranged in 1–3 rows along endostyle ( Fig. 2C). Ovaries facing atrial cavity; each ovary containing 10– 20 eggs( Fig. 2E). Testes against body wall; each testis consisting of two follicles ( Fig. 2E). Many endocarps uniformly present on body wall.   Etymology.This species is named after the typelocality, Sesoko Island. NH issued a call for research funding supported through the Japanese crowdfunding site “academist”, offering the opportunity to propose a name for this species as a token of gratitude for a major donor. The Destroyer Kikuzuki Association secured this right and requested that the species be named after its typelocality.  Phylogeny.Some degree of topological incongruence was observed between the phylogenetic trees generated by ML and BI.  Eusynstyelaseems to be non-monophyletic in the phylogenetic tree, although almost all branches have low support values ( Fig. 3). The phylogenetic analysis suggests that  E. misakiensis,  E. latericius( Sluiter, 1904), and  E. sesokoensis sp. nov.may form a clade and that  E. hartmeyeri Michaelsen, 1904and  E. tincta( Van Name, 1902)appear to be placed outside of this group.   Remarks.Due to taxonomic conflict regarding wheth- er  Eusynstyelashould be treated as a genus or a subgenus,  Eusynstyela misakiensishas been referred to as  Polyandrocarpa( Eusynstyela) misakiensis. In fields outside taxonomy, researchers have adopted  P.( E.) misakiensis, often referring to the species simply as  P. misakiensis. This may reflect either of two possibilities: that the subgenus name is simply omitted or that it is not recognized as valid. Although the support values in the phylogenetic tree are low, it is recommended that the species be designated to as  E. misakiensis, as the tree recovers a clade consisting of  E. latericius,  E. misakiensis, and  E. sesokoensis sp. nov., excluding  P. zorritensis( Van Name, 1931)( Fig. 3). A phylogenomic tree based on transcriptome data by Alié et al. (2018)also showed that  E. misakiensis(treated therein as  P. misakiensis) formed a clade with  E. tincta(the typespecies of  Eusynstyela), while  Polyandrocarpa zorritensisclustered together with  DendrodoaMacLeay, 1824and  Polycarpa. Denser taxon sampling of both  Eusynstyelaand  Polyandrocarpawill be necessary to refine molecular phylogenetic analyses; nevertheless, current evidence suggests that the two represent distinct genera. The new species can be regarded as a member of the genus  Eusynstyelabased on the following four morphological characteristics. First, the colonies consist of asexually reproduced zooids. Second, each side of a pharynx has four folds. Third, each gonad has two malefollicles; the specimens cannot be assigned to  Monandrocarpa, which has a single male follicle per gonad, or to  Polyandrocarpa, which possesses multiple per gonad. Fourth, the body wall has endocarps. Moreover, the phylogenetic analysis is consistent with this view; the new species was recovered as sister to  E. latericius, though the branch support is low ( Fig. 3).  Fig. 3. A maximum-likelihood tree based on the concatenated sequence of 18S (1839 bp) and COI (839 bp), indicating the position of  Eusynstyela sesokoensis sp. nov.within Styelidae. Numbers on branches showing ultrafast bootstrap values (≥50) and posterior probability (≥0.50). If only one value is shown at a branch, it represents the ultrafast bootstrap value. The most distinctive trait for differentiating  E. sesokoensis sp. nov.from its congeners is the absence of a stomach caecum ( Table 1). Within this genus, only  E. grandis Kott, 1990and  E. sesokoensis sp. nov.lack a stomach caecum. The morphological differences between these two species are as follows: i) number of tentacles ( 24 in  E. grandisvs. ten in  E. sesokoensis sp. nov.), ii) number of stomach plications ( 16 in  E. grandisvs. 12 in  E. sesokoensis sp. nov.), iii) number of lobes on the anal rim (eight in  E. grandisvs. zero in  E. sesokoensis sp. nov.), and iv) number of testes per gonad (one in  E. grandisvs. two in  E. sesokoensis sp. nov.). Additionally,  E. sesokoensis sp. nov.occasionally exhibits zooids with three gonadal rows on the right side ( Fig. 2C), whereas it is customary for zooids in this genus to have one or two gonadal rows ( Table 1). This unique feature further distinguishes  E. sesokoensis sp. nov.from its congeners.  An intraspecific variation in the coloration of zooids was noted, with the dorsal side appearing orange in the holotype( NCHSU 962) and the paratypes( NCHSU 963, 967, 968) or yellow in the other paratypes( NCHSU 964–966) ( Fig. 2A, B). The presence or absence of white spots in intraspecific variation of  E. misakiensisis determined by the distribution of red pigment in the epidermal layer, and this phenotype is genetically stable and not affected by environmental conditions ( Ishii et al. 1993). Although not confirmed, a similar mechanism perhaps involved in the coloration pattern observed in the new species. Within the pharynx on each side, the first and third folds from the dorsal lamina tended to have two to three times more longitudinal vessels than the second and fourth folds (Table 2). This tendency, however, was not observed in one of the paratypes, NCHSU 965(Table 2). No variation was observed among the specimens in the number of tentacles, pharyngeal folds, stomach plications, or male follicles per gonad. NCHSU 3 102 NCHSU 962 1 holotype NCHSU 3 102 NCHSU 963-968 2 paratype NCHSU Sesoko Island 3 102 NCHSU 963 2 paratype [156,633,849,873] NCHSU 3 102 NCHSU 964 1 paratype [650,1428,198,873] NCHSU Kushida of Rissho University 3 102 NCHSU 965-967 1 paratype [820,1321,231,255] NCHSU 3 102 NCHSU 968 1 paratype NCHSU 5 104 NCHSU 962, NCHSU 963, 967, 968, NCHSU 964-966 3 holotype NCHSU 5 104 NCHSU 965 1 paratype