Histology-free description and phylogenetics of Tetrastemma parallelos sp. nov. (Nemertea: Eumonostilifera) from Japan Author Abato, Jamael Author Yoshida, Ryuta Author Kajihara, Hiroshi text Journal of Natural History 2022 2022-09-19 56 29 - 32 1265 1277 http://dx.doi.org/10.1080/00222933.2022.2118642 journal article 10.1080/00222933.2022.2118642 1464-5262 7156370 7A50603A-9D73-449D-A2E7-343C20AF3A23 Tetrastemma parallelos sp. nov. ( Figure 1 (a–d)) Material examined Holotype : ICHUM 8300 , total DNA extracted from a single specimen dredged from a depth between 11 and 18 m on 2 December 2021 , off Kouyatsu , Tateyama , Chiba , Japan (between 34.988°N , 139.809°E and 34.984°N, 139.803°E). Diagnosis A Tetrastemma with a reddish cephalic patch and two orange, parallel dorsal stripes on a semi-transparent/translucent body. Description External features : The holotype specimen in the anaesthetised state is 2.6 mm long and 0.52, 0.65 and 0.60 mm wide at the anterior, mid and posterior body, respectively. Generally, the body is nearly flat, colourless except for some pigmented regions and marginally round at both ends. Dorsally, the head is spatulate in shape, narrower, and not completely demarcated from the body. The cephalic lobe is dotted with a quadrangular reddish patch somewhat restricted and concentrated at the centre of the cephalic lobe, in the anterior-eye region, leaving a transparent lobe margin. Two pairs of distinct, oblique lateral cephalic furrows, anterior and posterior, are present. The cephalic furrows in each pair do not join with one another. Two large, dark red anterior eyes are prominently visible within the pigmented cephalic region (above the anterior cephalic furrow) ( Figure 1 (a)). These anterior eyes appear to be almost black at higher magnification ( Figure 1 (c)). The two posterior eyes are situated near the posterior cephalic furrows, smaller than the anterior ones, and visible only ventrally ( Figure 1 (b)) or in squeezing preparation ( Figure 1 (c)). Dorsally, the body is characterised by the presence of two orange-pigmented parallel lines, which originate near the posterior cephalic furrows and are transversely connected to each other at their anterior ends. These orange lines run through the entire body: anteriorly, the stripes appear thinner; posteriorly, the stripes are thicker and begin to converge near the anus but never connect to each other ( Figure 1 (a)). Ventrally, the body is semi-transparent/translucent ( Figure 1 (b)). Figure 1. Tetrastemma parallelos sp. nov. , holotype ICHUM 8300: (a) anaesthetised specimen, dorsal view; (b) anaesthetised specimen, ventral view; (c) magnification of the head, squeezed specimen, dorsal view; (d) magnified stylet apparatus. Scale bars: a, b = 1 mm; c, d = 50 µm. Internal features : Dorsally, portions of the digestive system are visible through the transparent body as a wide, creamy to pale yellow region running along the body from below the neck area down to the posterior end ( Figure 1 (a)). Ventrally, an opaque-white region is notable between the cephalic patch and the posterior eyes. The rhynchocoel is visible and as long as the body. Lateral diverticula of the intestine are apparent; the anterior-most intestinal caecal diverticula extend behind the brain ( Figure 1 (c)). Gonads are very apparent ( Figure 1 (b)). Central stylet is 67.2 µm long. Stylet basis is 93.0 µm long and 55.5 µm wide at the most posterior part. It is nearly triangular, with darker, truncated posterior region and lighter, nearly acute anterior region. Two accessory-stylet pouches are visible, each containing 2–3 accessory stylets ( Figure 1 (d)). Etymology The new specific name is a noun in apposition (in the nominative case), derived from the Greek παράλληλος ( parallelos , ‘parallel’). The specific name pertains to the orangepigmented parallel lines on the dorsal body, which are one of the diagnostic features of the new species. Type locality and distribution At present, the species is only known from its type locality, off Kouyatsu , Tateyema , Chiba Japan , 11–18 m depth , mudstone and sandy bottom ( Figure 2 ); the in situ water temperature was 18–20°C when the holotype was collected . Molecular phylogeny The phylogenetic analyses confirmed that Tetrastemma parallelos sp. nov. belongs to the Asian–Australian Pacific Tetrastemma subclade. Our species was more closely related to those Tetrastemma species labelled as VE Vietnam , VI Vietnam and 1G4 Australia (in Chernyshev et al. 2021 ) than to the remaining species included in the analyses ( Figure 3 ). Remarks Several Tetrastemma species are known to have one or more longitudinal dorsal stripes. In having two parallel dorsal stripes, Tetrastemma parallelos sp. nov. resembles some congeners such as Tetrastemma bilineatum Coe, 1904 , Tetrastemma bistriatum ( Timofeev, 1911 ) , Tetrastemma herthae Corrêa, 1963 , Tetrastemma nigrolineatum Wheeler, 1934 and Tetrastemma scutelliferum Bürger, 1895 , as well as some varieties of Tetrastemma nigrifrons Coe, 1904 including bilineatum ( Iwata 1954 ) , bimaculatum ( Chernyshev 1998 ) (see also Zaslavskaya et al. 2010 ) and trimaculatum ( Chernyshev 1998 ). Figure 2. Conditions of the bottom surface where Tetrastemma parallelos sp. nov. was collected. Figure 3. Rooted Bayesian phylogenetic tree showing the position of Tetrastemma parallelos sp. nov. within the Tetrastemma Asian–Australian Pacific subclade ( Chernyshev et al. 2021 ) based on concatenated sequences of five gene markers (16S, 18S, 28S, COI and H3). Numbers near each node indicate Bayesian posterior probability (PP)/RAxML maximum-likelihood bootstrap value (BS)/IQTree maximum-likelihood ultrafast bootstrap value (UFBoot). Solid circles indicate full support (1.00 PP/ 100% BS/100% UFBoot). Open circles indicate 1.00 PP. In T. bilineatum , the dorsal twin stripes are reddish brown to deep chocolate in colouration. Anteriorly, the stripes terminate somewhat in front of the ocelli but sometimes reach the very tip of the snout. Posteriorly, they extend nearly, though not quite, to the posterior extremity of the body and are sharp and conspicuous throughout. Anteriorly, these stripes are not transversely connected with one another but converge at the tail region ( Coe 1904 ). Additionally, this species lacks a cephalic patch. Tetrastemma bistriatum has two dark brown stripes running dorsally that are separated by a wide area darker than the rest of the body. A dark-brown half-ring is present behind the posterior eyes, from which the two dorsal stripes extend ( Timofeev 1911 ). Dorsally, T. herthae has two lateral longitudinal stripes strongly conspicuous for their deep brown colour. Sometimes they are not continuous but appear as rows of brown irregular spots placed closely together. The stripes do not join at the ends ( Corrêa 1963 ). Tetrastemma nigrolineatum is characterised by two parallel black lines passing dorsally from the tip of the snout to the tip of the tail. At the head, these lines are thinner than they are in the body ( Wheeler 1934 ). Tetrastemma scutelliferum has two wide, dark brown longitudinal dorsal stripes, which begin behind the cephalic region and almost reach the hind end of the body. Also, the two dark brown stripes are separated by a broad yellow stripe which similarly extends near the posterior end. It also has a dark brown, lobular cephalic patch, which is divided into four lobules: two large lobes at the front and two smaller ones at the sides ( Bürger 1895 ). The three varieties of Tetrastemma nigrifrons commonly have brown or dark brown dorsal stripes, with varied colour and shape of the cephalic patch (dark brown, non-split cephalic patch in var. bilineatum ; two dark brown, split cephalic patches in var. bimaculatum ; and three dark brown, split cephalic patches in var. trimaculatum ) ( Iwata 1954 ; Chernyshev 1998 ; Zaslavskaya et al. 2010 ). Tetrastemma parallelos sp. nov. can be differentiated from these Tetrastemma species with two dorsal stripes by its orange-pigmented parallel lines transversely connected anteriorly and by its reddish cephalic patch. Although T. parallelos sp. nov. is smaller, the general body shape and the dorsal body feature of our species likely resemble those of T. bistriatum and T. scutelliferum . Our species is similar to T. bistriatum as the latter’s dorsal stripes appear to be connected anteriorly by way of its dark-brown half-ring. Internally, the morphology of the stylet basis differs between the two. The basis in T. bistriatum is rounded at the posterior end, unlike the truncated one in T. parallelos sp. nov. Aside from the slight dorsal-body feature resemblance, our species, like T. scutelliferum , has two accessory-stylet pouches. However, T. parallelos sp. nov. has only 2–3 accessory stylets in each pouch, compared with four accessory stylets per pouch in T. scutelliferum . In the resulting trees, T. parallelos sp. nov. was more closely related to the three undescribed forms in Chernyshev et al. (2021) – Tetrastemma sp. VE Vietnam , Tetrastemma sp. VI Vietnam , and Tetrastemma sp. 1 G4 Australia – than to other congeners ( Figure 2 ). However, T. parallelos sp. nov. can be distinguished from these three by the presence of the reddish cephalic patch and dorsal stripes. In addition, our species is shorter in body size than Tetrastemma sp. VI Vietnam ( 8–12 mm long) and VE Vietnam ( 8 mm long); and it has a semi-transparent/translucent body compared to the pale yellowish-orange and pale yellow body colouration of Tetrastemma sp. VI Vietnam and Tetrastemma sp. VE Vietnam , respectively. In this study, the histology-free approach was successful because (1) the assessment of genus affiliation was facilitated by molecular phylogenetics and (2) the new species obviously differed from all congeners in external appearance. If the new species had nested in a clade to which no generic name was reliably applied, its generic affiliation would have had to be assessed by traditional histology. Our enumeration indicates that there are ~40 eumonostiliferous hoplonemertean genera that are not yet represented by any molecular data; until all of these generic names are molecularly tagged, histological examination will continue to be indispensable in eumonostiliferous hoplonemertean systematics. In Tetrastemma , body colouration and patterns have been considered effective morphological characters to delimit species ( Strand and Sundberg 2005b ). Meanwhile, two or more closely related, similar-looking species of eumonostiliferous hoplonemerteans can be distinguished by subtle differences such as the epidermal texture (Abato pers. obs.), which would have not been noticed and verbalised without the use of a high-quality digital camera. This indicates that external features of all the Tetrastemma species in living state – no matter if the species is already described or not – should be photographed at high resolution, so that future histology-free descriptions continue to be effective.