﻿Three new species in Tetrastemma Ehrenberg, 1828 (Nemertea, Monostilifera) from sublittoral to upper bathyal zones of the northwestern Pacific

﻿Abstract Monostiliferous nemerteans in the genus Tetrastemma Ehrenberg, 1828 are generally characterized as having four eyes, and they occur worldwide, from the intertidal zone to the deep-sea bottom. Recent extensive sampling of Tetrastemma has explored the high species diversity, including many undescribed forms, but phylogenic analysis has revealed non-monophyly of the genus. We herein describe three new species of the genus (T.albumsp. nov., T.personasp. nov., and T.shohoensesp. nov.) from northwestern Pacific waters based on specimens collected by dredging or by use of a remotely operated vehicle at depths of 116–455 m. Since anatomical and histological characters traditionally used in systematics of the genus are sometimes interspecifically uniform, a histology-free approach is applied for the species descriptions in this study. To confirm the generic affiliation of the new species, a molecular phylogenetic analysis based on partial sequences of cytochrome c oxidase subunit I, 16S rRNA, 18S rRNA, 28S rRNA, and histone H3 genes was performed. Our result shows that all three new species are nested in a subclade formed by species from the North Pacific and American Atlantic, inferring that geographic distribution does not reflect the cladogenesis of Tetrastemma. Furthermore, two Tetrastemma species with a cylindrical stylet basis, T.freyaeChernyshev et al., 2020 from off the coast of India and Hawaii and T.shohoensesp. nov. from Shoho Seamount, Japan, constitute a clade in the resulting tree.


Introduction
A histology-free description with DNA barcoding has been progressively introduced to nemertean systematics in the past decade (e.g., Kajihara 2015;Gonzalez-Cueto et al. 2017;Simpson et al. 2017;Kajihara et al. 2018Kajihara et al. , 2022Chernyshev et al. 2020;Hookabe et al. 2021a, b;Leiva et al. 2021;Abato et al. 2022). This approach has been applied to two cases, one of which is a description of species with internal characters interspecifically differentiated and observable without histology (e.g., number of proboscis branches in Gorgonorhynchus Dakin & Fordham, 1931 [Kajihara 2015;Hookabe et al. 2021a)]. In the other case, especially when internal morphology is uniform between most species in a genus, a species description has been performed solely based on characters examined in-vivo (shape of head, body coloration and markings, number of eyes, blood color, and stylet apparatus) [e.g., Baseodiscus Diesing, 1850 and Ototyphlonemertes Diesing, 1863(Kajihara et al. 2018]. Recent descriptions of species in the genus Tetrastemma Ehrenberg, 1828, fitting the latter case, have been performed based on characters of living specimens without histological observations (Chernyshev et al. 2020;Hookabe et al. 2021b;Abato et al. 2022).
Tetrastemma is a species-rich genus in Monostilifera (Kajihara 2021), currently encompassing about 110 species from tropical to polar areas (Chernyshev et al. 2021). As the generic name suggests-a composite of the Latin feminine "tetra" (= four) + "stemma" (= simple eyes)-members in the genus are generally characterized by four eyes, but this feature is also found in other genera. Several species in Tetrastemma were described based on internal morphology; however, the internal characters were inferred to be almost homogenous within the genus by taxonomic reappraisal based on molecular phylogeny (Chernyshev et al. 2021). Recent examples of a histology-free approach based on characteristics studied in-vivo and molecular data are descriptions of T. freyae Chernyshev et al., 2020, T. cupido Hookabe, Kohtsuka & Kajihara, 2021, and T. parallelos Abato, Yoshida & Kajihara, 2022 Here, we establish three new species based on specimens collected in 2019-2021 from the lower sublittoral to upper bathyal zones of Sagami Bay and the Nishi-Shichito Ridge. The descriptions are histology-free, based on characters of living specimens examined with a light microscope. To test phylogenetic relationships with the congeners, we performed molecular phylogenetic reconstruction using partial sequences of the 16S rRNA (16S), cytochrome c oxidase subunit I (COI), 18S rRNA (18S), 28S rRNA (28S), and histone H3 genes (H3).

Materials and methods
Specimens were collected in 2019-2021 by use of a biological dredge in Sagami Bay (116-200 m) or a remotely operated vehicle (ROV) on Shoho Seamount of the Nishi-Shichito Ridge (455 m), northwestern Pacific Ocean. External morphology of the living specimens was documented on the vessel or in the laboratory with a Nikon D5600 digital SLR camera equipped with an AF-S DX Micro-NIKKOR 40mm f/2.8G macro lens (Nikon, Japan). A single specimen collected from Shoho Seamount was further observed under a compound light microscope by preparing a squeezed specimen with a cover slip and a glass slide. Specimens were anaesthetized with a few drops of bitterns Tenpi Nigari (Amashio, Japan); after the worms were relaxed, the posterior tips were preserved in 99% ethanol for DNA extraction and the rest of the body was fixed in Bouin's fluid for 24-48 hours and later transferred to 70% ethanol. Type specimens have been deposited in the National Museum of Nature and Science, Tsukuba (NSMT), Japan.
DNA extraction, PCR amplification, and sequencing followed Hookabe et al. (2022). DNA sequences determined in the present study have been deposited in DDBJ/EMBL/GenBank (Table 1). To elucidate phylogenetic positions of specimens examined, we performed phylogenetic analyses based on the maximum-likelihood (ML) method. The newly obtained sequences from four Tetrastemma species were aligned using MAFFT v. 7 (Katoh and Standley 2013) employing L-INS-i strategy with sequences of other species in the genus, most of which were recently determined by Chernyshev et al. (2021). Ambiguous nucleotide sites in the dataset were removed with Gblocks v. 0.91b (Castresana 2000) using a less stringent option, resulting in 380-bp 16S, 626bp COI, 1738-bp 18S, 505-bp 28S, and 329-bp H3. The ML analyses were performed with RAxML-NG (Kozlov et al. 2019), for which the best-fit partition scheme and substitution model were selected using PartitionFinder v. 2.1.1 (Lanfear et al. 2017).
Nodal support values were derived from 1000 bootstrap pseudoreplicates.

Genus Tetrastemma Ehrenberg, 1828
Tetrastemma album sp. nov. https://zoobank.org/F73378DB-B867-4ABA-A2D7-18CF781D10A7 Fig Description. Head spatulate to rounded in profile ( Fig. 2A-C), demarcated by posterior cephalic furrows from body ( Fig. 2A). Before anesthetization, body of a live specimen 17 mm long and 1.0-1.2 mm wide. Body uniformly pale colored, without longitudinal or transverse stripe markings ( Fig. 2A). Pure white transverse cephalic patch present between anterior and posterior pairs of eyes (Fig. 2B). Head not wider than maximum body width ( Fig. 2A-C). A pair of cephalic furrows present; anterior pair not meeting mid-dorsally and ventrally curving anteriorly but not reaching to proboscis pore; posterior pair V-shaped and barely meeting mid-dorsally (Fig. 2B) and running transversely on ventral surface (Fig. 2C). Cerebral ganglia and blood not red and probably uncolored. Internal organs (proboscis, foregut, and intestine) visible as pale regions. Four reddish brown eyes regular in size (Fig. 2B). Type locality and distribution. The species is only known from the type locality, Sagami Bay, Kanagawa Prefecture, Japan, at depths of 144-200 m (Fig. 1).
Remarks. Having a pure white cephalic patch on a uniformly pale body, T. album sp. nov. differs from all the described species. Tetrastemma coronatum (Quatrefages, 1846), T. diadema Hubrecht, 1879, T. olgarum Chernyshev 1998, and T. pseudocoronatum Chernyshev 1998 have white cephalic patches but are distinguished from T. album sp. nov. in possessing a light brown to dark transverse band on the head. Tetrastemma albomaculatum Chernyshev, 2016 also possesses a white cephalic patch but differs from the new species in having a pale-ochre body dorsally spotted with small white dots (Chernyshev 2016).

Tetrastemma persona sp. nov.
https://zoobank.org/E3E48065-E551-477D-9041-89DEE011DEB0 Fig. 2D [New Japanese name: misaki-kamen-himomushi] Etymology. The species name is derived from the Latin persōna (mask), referring to a broad cephalic patch of the new species masking eyes and internal organs in head region. The Japanese name "kamen" means a mask in Japanese.
Description. Head slightly narrower than middle part of body and weakly demarcated from trunk (Fig. 2D). Before anesthetization, body of a live specimen 7.0-10 mm long and 0.8-1.0 mm wide. Body uniformly pale to yellow colored without longitudinal or transverse stripe markings (Fig. 2D). Vermilion-red cephalic patch spade-shaped (Fig. 2D), covering both anterior and posterior pairs of eyes ( Fig. 2D) but not posteriorly reaching to anterior pair of cephalic furrows; eyes regular in sizes. A posterior pair of cephalic furrows not well distinguished probably due to the small body size. Cerebral ganglia and blood not red and probably uncolored. Internal organs (proboscis, foregut, and intestine) not well visible through body wall. Rhynchocoel visible as whitish region through body wall, extending about 1/2-2/3 of the body length.
Type locality and distribution. The species is only known from the type locality, Sagami Bay, Kanagawa Prefecture, Japan, at depths of 116-211 m (Fig. 1, Table 1).
Remarks. Tetrastemma persona sp. nov. has atypically short rhynchocoel in the genus and most resembles T. roseocephalum (Yamaoka, 1947) and T. yamaokai Iwata, 1954 in having a pale body without any markings and a red cephalic patch. Pattern variation of a cephalic patch (shield shape or horse-shoe shape) was reported in both T. roseocephalum and T. yamaokai; referring to the original description of T. yamaokai, the name may be a junior synonym of T. roseocephalum, as suggested by Kajihara (2007). The external morphology of T. persona sp. nov. is similar to a form with a shield-shaped cephalic patch of T. roseocephalum (Iwata 1954).
The subtle difference in the shape of cephalic patch between T. persona sp. nov. (spade-shaped) and T. roseocephalum (shield-shaped) was supported by our molecular analysis. The new species did not constitute a clade with T. roseocephalum but with T. album sp. nov. (Fig. 3); T. roseocephalum belongs to Clade C of Chernyshev et al. (2021).
An uncorrected genetic distance based on 657 bp of COI was 16% between T. album sp. nov. and T. persona sp. nov., comparable with interspecific values observed among Monostilifera (e.g., Sundberg et al. 2016;Hookabe et al. 2022). Description. Head spatulate in profile (Fig. 2E-H), not well demarcated from body by anterior cephalic furrows (Fig. 2E). Before anesthetization, body of a live specimen 5.5 mm long and 0.3 mm wide. Background body color generally white, tinged with bright yellow to orange, and almost transparent (Fig. 2E). Head with a red rectangle cephalic patch without extending behind a posterior pair of eyes (Fig. 2E, H). Anterior pair of cephalic furrows present (Fig. 2F) but posterior one not well distinguished. Cerebral ganglia and blood uncolored (Fig. 2G, H). Alimentary canals visible as bright yellow organs through body wall (Fig. 2E). Proboscis pale, extending about 3/4 of the body length (Fig. 2E). Four brown eyes present; anterior pair slightly larger than posterior ones (Fig. 2H).
Type locality and distribution. The species is only known from the type locality, Shoho Seamount of the Nishi-Shichito Ridge, Japan, at a depth of 455 m (Fig. 1), among the sandy sediments on rocky substrates.
Remarks. Having a dark cephalic patch and cylindrical stylet basis and lacking a longitudinal line on the dorsal surface of the body, T. shohoense sp. nov. resembles T. freyae Chernyshev et al., 2020 originally described based on Hawaiian and Indian specimens. The new species is differentiated from T. freyae in the color of the cephalic patch as well as the non-flared posterior margin of the cylindrical stylet basis.

Molecular phylogeny
The sequence data set for molecular phylogenetic analyses in the present study is primarily based on Chernyshev et al. (2021). Since we confirmed that our new species are nested in Tetrastemma Clade B of Chernyshev et al. (2021), we used three species in Clade A (Tetrastemma sp. GM1 Gulf of Mexico, Tetrastemma sp. GM2 USA FL, and Tetrastemma sp. GM3 Gulf of Mexico) as outgroup taxa (Fig. 3). Clade B was subdivided into two clades with a full support value, one of which was a clade formed by T. freyae and T. shohoense sp. nov. The two species are characterized by having a cylindrical stylet basis in the proboscis. In the other subclade in Clade B, T. album sp. nov. and T. persona sp. nov. were included (Fig. 3). A clade constituted by newly described  species, T. album sp. nov. and T. persona sp. nov., from Sagami Bay (Japan) with 99% of BS, was nested in the American Atlantic clade formed by T. elegans (Girard, 1852) (Virginia), T. enteroplecta (Corrêa, 1954) (Florida and Venezuela), T. merulum (Corrêa, 1954) (Florida), and Tetrastemma sp. F7 (Florida). The clade formed by T. album sp. nov. and T. persona sp. nov. was sister-related to a clade formed by T. enteroplecta (Florida and Venezuela) and Tetrastemma sp. F7 (Florida) with 77% of BS (Fig. 3).

Discussion
Three species herein described (T. album sp. nov., T. persona sp. nov., and T. shohoense sp. nov.) fell within a clade referred to as Tetrastemma Clade B of Chernyshev et al. (2021) (Fig. 3). One of the findings from the tree is that two species with cylindrical stylet basis, T. freyae and T. shohoense sp. nov., formed a clade regardless of the differences in habitat and collection depths of these two species; T. freyae was described based on specimens collected from live corals and mussel beds at depths shallower than 3 m in Hawaii and India (Chernyshev et al. 2020), while T. shohoense sp. nov. was found from sandy sediments in bathyal zone in Japan. A cylindrical stylet basis is likely to be acquired independently at least twice in Clade B (T. freyae and T. shohoense sp. nov.) and Clade C (T. albomaculatum and T. parallelos).
The other thing we can see on the phylogenetic tree is that T. album sp. nov. and T. persona sp. nov. are nested in a clade formed by several American Atlantic species, T. enteroplecta, T. elegans, T. merulum, and Tetrastemma sp. F7 (Chernyshev et al. 2021) (Fig. 3). A previous molecular analysis has inferred that Tetrastema clade B is subdivided into geographically distinct structures: North Pacific and American Atlantic subclades (Chernyshev et al. 2021). To obtain a more accurate picture of Tetrastemma phylogeny and speciation, again, further sampling of taxa, without bias toward shallow-water species, is needed for future phylogenetic analyses.