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CTENOPHORICOLA MASANORII SP. NOV. Figs 2–7, Supporting Information (Movies S1–S3) lsid: zoobank.org:act: 4E29046B-899F-410C-8A46- C1E99F5E50E9 Material examined: Holotype: MNCN 16.01 /17896, off south-east Enoshima Island (Kanagawa Prefecture, Japan) on Beroe campana Komai, 1918, fixed in 4% formaldehyde buffered in seawater, preserved in 70% ethanol, 9 September 2013. Paratypes: MCZ 25325 & 25326 (two adults) and MNCN 16.01 /17894 (two juveniles), on Bolinopsis mikado Moser, 1908, 96% ethanol; MNCN 16.01 /17895 (six adults, one juvenile) on Bo. mikado, fixed in 4% formaldehyde buffered with seawater, preserved in 70% ethanol; MNCNM 16.01 /17898 (six adults, three juveniles) on Be. campana, 96% ethanol; MNCN 16.01 /17897 (four adults, two juveniles) and MNCN 16.01 /15342 (two additional adults used for SEM), fixed in 4% formaldehyde buffered with seawater. All collected off south-east Enoshima Island, Kanagawa Prefecture, Japan, 9 September 2013. Diagnosis: Species of Ctenophoricola with transverse pigmented bands, lightly coloured on dorsum and lacking marked caeca in gut. Description of largest individuals (adults?): Holotype (Figs 2A, B) 2.6 mm long (live, relaxed specimens up to 3 mm long), anterior and posterior regions 0.4 mm and 0.6 mm wide, respectively, excluding parapodia. Body small, cylindrical, somewhat dorsoventrally flattened on posterior half, with peristomium and 27 segments with parapodia, divided in two distinctly different regions (Figs 2A, 3A, 5 C-D), densely covered by short cilia (Figs 2 B-C, 3, 4). Translucent to yellowish in vivo (Fig. 5), with one reddish narrow transverse band dorsally on peristomium, sometimes with a pigmented band on each of the two most anterior segments (Fig. 2A, C) and on posterior region (Fig. 2A) segments; some reddish areas on laterals of some segments; other specimens without colour pattern (Fig. 5). Prostomium small, semicircular, without external eyes, with two minute, retractile palps distally ciliated, partially retracted inside peristomium (Figs 2A–C, 3B–C, 4B–D). Peristomium similar in length to subsequent segments, but narrower than chaetigers; a median, small, dorsal lobe on some specimens (Figs 2A, C, 4 B-C) often with two minute papillae (Figs 2A, C, 3B, 4D). Mouth covered by one dorsal and two ventral lips (Fig. 3 B-C). Pharynx cylindrical, everted in some specimens (Fig. 4B, D), unarmed, without papillae. Two conspicuous, anterior, interior lensed eyes, either dark reddish-brown or yellowish (e.g. in juveniles) (Fig. 5), internally reaching to segment 4–5 in preserved specimens (2–3 in vivo). Anterior region with 12–13 segments. Parapodia conical, with thin, internal acicula, without chaetae (Figs 2, 3A–D, 4B, D-E), with a small, indistinct, rounded, dorsal lobe, densely covered by cilia (Fig. 4E); ventral lobe similar (Fig. 3D); most anterior parapodia laterofrontally directed (Figs 2, 4B, D). Posterior region with 15 segments, distinctly wider than those of anterior region; sometimes with distinct clusters of large cells in larger individuals (Fig. 2A); parapodia larger and longer with internal slender acicula and a fascicle of few, thin capillary chaetae; dorsal and ventral lobes distinctly enlarged (Figs 2A, 3A, E-F, 4A) and lateroposteriorly directed. Gut straight, visible through body wall, distinctly wider in posterior region (Figs 1A, 5 C-D). Pygidium small with two rounded, short anal cirri. Description of smallest individuals: Minute specimens (c. 0.33 mm long), showing slightly bilobed prostomium, non-perceptible palps and a welldefined peristomium, with no sign of peristomial cirri. Anterior region with c. seven segments, with parapodia similar to those of adults; posterior region as a triangular bud showing traces of segments, but lacking parapodia. Gut is fully developed. External eyes not detectable (Fig. 6A). Specimens of c. 0.5 mm are similar to the samallest ones (i.e. 0.33 mm), with a more developed posterior region and distinct segments, but still lacking parapodia and traces of internal eyes (Figs 6B, 7 middle). Specimens of c. 1.2 mm, are more similar to the largest ones (adults?), with well-developed posterior parapodia, and yellowish internal eyes, but with posterior region (Figs 6C, 7 right, Supporting Information, Movie S3) not as distinct as in the largest specimens (adults?) parapodia. Behaviour: Ctenophoricola masanorii were observed feeding directly on the surface of the host by everting their pharynx and sucking from the epithelia and underlying mesoglea. They triggered a contraction of the affected area (Supporting Information, Movie S3) similar to that caused by the movement of other parasites (Supporting Information, Movie S2), suggesting they disturb the host. Locomotion of C. masanorii results from accordion-like contractions and extensions in which the anterior region extends, attaches to the ctenophore surface by parapodia and then is slowly contracted pulling the posterior region forward (Supporting Information, Movies S1 and S3). The worms were observed extending the anterior region out away from the host while the posterior region was still attached (Supporting Information, Movies S1 and S3), either to explore all of the available host surface before choosing a direction to head in or to find new hosts. Three specimens that may be regenerating or juvenile and subadult forms, appeared on the same ctenophore as adults (Figs 6–7); early (Figs 6A, 7 left) and later (Fig. 6B, 7 middle) regenerating/juveniles were found together with a specimen almost identical to the adult (i.e. subadult, Figs 6C, 7 right, Supporting Information, Movie S2). These small specimens seemed to have limited capacities either to maintain their attachment to the host or to float in the water column (Supporting Information, Movie S2). Thus, we suggest that some of these small specimens are able to hold on the same as the largest forms, whereas others detach and drift either until they encounter another host or they die in the water column. Type locality: South-east Enoshima Island, Japan. Habitat and distribution: On the surface of the ctenophores Bo. mikado and Be. campana. Known only from the type locality in Japan. Etymology: The species is named after Dr Masanori Sato, from Kagoshima University, a well-known and enthusiastic polychaetologist, who sent the specimens of the new species, together with the pictures and videos, and also provided the co-first authors with numerous and valuable syllids from Japan., Published as part of Martín, Guillermo San, Álvarez-Campos, Patricia, Kondo, Yusuke, Núñez, Jorge, Fernández-Álamo, María Ana, Pleijel, Fredrik, Goetz, Freya E., Nygren, Arne & Osborn, Karen, 2021, New symbiotic association in marine annelids: ectoparasites of comb jellies in Zoological Journal of the Linnean Society 191

Annelids are a broadly distributed, highly diverse, economically and environmentally important group of animals. Most species can regenerate missing body parts, and many are able to reproduce asexually. Therefore, many annelids can generate all adult cell types in adult stages. However, the putative adult stem cell populations involved in these processes, as well as the diversity of adult cell types generated by them, are still unknown. Here, we recover 75,218 single cell transcriptomes of Pristina leidyi, a highly regenerative and asexually-reproducing freshwater annelid. We characterise all major annelid adult cell types, and validate many of our observations by HCR in situ hybridisation. Our results uncover complex patterns of regionally expressed genes in the annelid gut, as well as neuronal, muscle and epidermal specific genes. We also characterise annelid-specific cell types such as the chaetal sacs and globin + cells, and novel cell types of enigmatic affinity, including a vigilin + cell type, a lumbrokinase + cell type, and a diverse set of metabolic cells. Moreover, we characterise transcription factors and gene networks that are expressed specifically in these populations. Finally, we uncover a broadly abundant cluster of putative stem cells with a pluripotent signature. This population expresses well-known stem cell markers such as vasa , piwi and nanos homologues, but also shows heterogeneous expression of differentiated cell markers and their transcription factors. In these piwi + cells, we also find conserved expression of pluripotency regulators, including multiple chromatin remodelling and epigenetic factors. Finally, lineage reconstruction analyses reveal the existence of differentiation trajectories from piwi + cells to diverse adult types. Our data reveal the cell type diversity of adult annelids for the first time and serve as a resource for studying annelid cell types and their evolution. On the other hand, our characterisation of a piwi + cell population with a pluripotent stem cell signature will serve as a platform for the study of annelid stem cells and their role in regeneration., Competing Interests: Competing Interests The authors declare that they have no competing interests.