The 18S and 28S sequences of Ramisyllis from Australia and Japan differed considerably from those of all other syllids. 18S showed insertions in regions V2 and V5 (which were especially difficult to align), while for 28S there are still few available sequences. Moreover, only our 28S sequences (both for Ramisyllis and T. cryptica) are complete, while most others available in Genbank represent only a short region (300–500 bp).
All COI, 16S, 18S, and 28S trees (Fig. 2), as well as those from the concatenated data matrix (COI + 16S + 18S + 28S) (Fig. 3) were congruent. In the COI, 16S and 28S ML tree inference analyses, the sequences of Ramisyllis (seven from Australia and seven from Japan) are organized in two well-supported sister clades (Figs. 2 and 3), with some structure within each clade being detected in COI and 16S (Fig. 2c, d) and no clear within-clade differences for 28S (Fig. 2b). In 16S, 28S, and, especially, COI, the branch length of each clade is long enough to recognize the Australian and Japanese groups (Fig. 2b–d), while in 18S, the within-clade branches are extremely short and the Japanese clade is nested within the Australian one, revealing few variations (Fig. 2a). In contrast, the branch length joining Ramisyllis with its sister group Trypanobia-Trypanedenta is considerably long in 18S (Fig. 2a) and 28S (Fig. 2b), whereas it is average when compared to other branch lengths within Syllinae for 16S and COI (Fig. 2c, d).
The Ramisyllis clade is nested within the “ribbon clade” (sensu Aguado et al., 2015a) in all inferred phylogenies and appears as sister group to Trypanobia and Trypanedenta clade (83B) in the concatenated data analysis (Fig. 3). The “ribbon clade” is always well supported but, in the concatenated data analysis, it shows an early subdivision in a well-supported (99B) small clade (Pseudosyllis brevipennis Grube, 1863 specimens) and a less-supported (64B) large clade including the remaining genera and species. Within this one, a large clade (67B) shows a dichotomy between the Xenosyllis-Plakosyllis-Eurysyllis clade (85B) and Trypanosyllis-Parahaplosyllis (98B). Eurysyllis, Xenosyllis and Parahaplosyllis, all represented by more than one species, are monophyletic, whereas Trypanosyllis appears as paraphyletic.
The complete mitochondrial genome of the Japanese Ramisyllis SA1 (Tables 1, S1) was identified in a single contig among the sequences of the assembled low-coverage genome. It is 16.517 bp long (longer than in R. multicaudata) and AT-rich (67%). A is the most common base (34%), and G the least common (12%). The coding strand has a strong skew of G vs. C (− 0.313), whereas the AT skew is positive (0.034). The mt-genome contains 37 genes (13 protein-coding genes, two rRNA genes, and 22 tRNA genes) as in most other annelids and typically present in bilaterian mt-genomes (Online Resource 3).
Gene arrangement did not differ between the Japanese and Australian Ramisyllis (Aguado et al., 2015a) (Fig. 4). The mt-genome of the Japanese Ramisyllis has a 821 bp long putative control region flanked by nad6 and trnL1 and 25 non-coding regions ranging from one to 320 bp, with the largest one between trnT and rrnD (Fig. 14; Online Resource 3). ATG and TAA are the start and stop codons for all 13 protein coding genes (Online Resource 3). There is also a marked codon usage bias (Online Resource 4), with NNGs being the least used, and NNTs and, especially NNAs being the most common. The ribosomal RNAs are 1011 bp long for 16S (rrnL), and 795 bp long for 12S (rrnS). The two genes are only separated by an intergenic spacer of 20 bp (Online Resource 3). As in the Australian Ramisyllis (Aguado et al., 2015a), the DHV stem is missing in trnC and trnR, while shortened in trnS1. In contrast, while the DHV stem in trnS2 is shortened in R. multicaudata from Australia, it is longer in Ramisyllis from Japan.
COI genetic distances between Australian and Japanese Ramisyllis are 20–21% based on a Tajima-Nei model and 17–18% based on p-distances (Online Resource 5), while the within-clade divergence is 1% and that between the Ramisyllis clade and other species of the “ribbon clade” range between 26 and 40% (Online Resource 6). Several species of Trypanosyllis show distances of 17–26% between them (in yellow in Online Resource 6). Trypanedenta gemmipara (Johnson, 1901), Trypanedenta gigantea (McIntosh, 1885), and Trypanobia asterobia (Okada, 1933) show distances of 16% among them. The 16S genetic distance between the Australian and Japanese Ramisyllis ranges between 10 and 11% based on Tajima-Nei model (Online Resource 7), while it is 0–1% within-clades. Distances obtained when analyzing the alignment including only Ramisyllis sequences (not shown) are the same as those obtained in the alignment with the rest of the “ribbon clade” species (Online Resource 7). The distance with other species in the ribbon clade ranges between 25 and 33% and some species of Trypanosyllis show distances of 11–15% (in yellow in Online Resource 7). In 28S, the genetic distance between the Australian and Japanese specimens is 4% (Online Resource 8), while in 18S, it is 1% (Online Resource 9), being the latter non-significant. In ITS2, the genetic distance between the Australian and Japanese specimens is 11% (Online Resource 10).
Ramisyllis Glasby et al., 2012
Diagnosis (after Glasby et al. (2012), emendations in bold).
“Ribbon clade” Syllinae, with non-flattened body, more or less cylindrical segments and a multiaxial, dendriform pattern; first branch occurring after segments 14–24. Branches emerging after parapodia (not replacing them or dorsal cirri) and showing same segment size and cirri length as previous branches. Three antennae; palps free to base; two pairs of tentacular cirri; pharynx slender, mid-dorsal tooth absent in adults; dorsal cirri articulated, with alternating thick/slender pattern on mid-body and posterior segments; ventral cirri present, not articulated, inserted proximally; single type of simple chaeta present, tomahawk-shaped. Sexes separate. Reproduction by schizogamy, gemmiparitity. Acerous, dimorphic stolons. Commensal inside shallow water species of Petrosia. Mitochondrial gene order strongly modified. Nuclear ribosomal sequences highly derived compared to other Syllinae.
n. sp. Aguado, Ponz-Segrelles, Glasby, Ribeiro, Jimi & Miura. Figures
Species of Ramisyllis, sister-group related to R. multicaudata, long anterior tentacular and dorsal cirri (twice long as midbody ones), long proventricle (through 4 segments), stolon stalks similar to other segments in regular branches and proliferation of new branches in intersegmental areas.
HOLOTYPE: Female (ZMUG 29,568, MNCNM 16.01/19089–90), samples SA1-SA6, 1 Oct 2019, 37°48′17.1″N, 138°14′25.1″E, 15 m deep, coll. Aguado, Ponz-Segrelles, Miura, Oguchi, Omori & Kohtsuka.
PARATYPES: Paratype 1: male (ZMUG 29,569, MNCNM 16.01/19091–92), samples SA7-22; paratype 2: male (ZMUG 29,571), samples SA28-34; paratype 3: non reproductive specimen (ZMUG 29,573, MNCNM 16.01/19093), samples SA43, 46; paratype 4: male (ZMUG 29,576), samples SA61-62; paratype 5: non reproductive specimen (ZMUG 29,576), sample SA63; paratype 6: male (ZMUG 29,583, MNCNM 16.01/19094–96), sample SA85-91; paratype 7: non reproductive specimen (ZMUG 29,585), sample 96; paratype 8: male (ZMUG 29,586), sample SA102; paratype 9: female (NSMT Pol-P-843), sample 220. All paratypes collected on 1st Oct 2019, 37°48′17.1″N, 138°14′25.1″E, 15 m deep, by Aguado, Ponz-Segrelles, Miura, Oguchi, Omori & Kohtsuka.
Additional material: 1 female (ZMUG 29,570), 1 male (ZMUG 29,572), 1 female (ZMUG 29,574), 1 male (ZMUG 29,578), 1 non reproductive specimen (ZMUG 29,579), 1 male (ZMUG 29,578), 1 female (ZMUG 29,582), 1 male (ZMUG 29,584), 1 male (ZMUG 29,589), 6 specs. (Sex not determined, undissected sponges with worms inside) (ZMUG 29,587, 29,590–94), male stolons (ZMUG 29,595, 29,598), female stolons (ZMUG 29,597). All specimens collected on 1st Oct 2019, 37°48′17.1″N, 138°14′25.1″E, 15 m deep, by Aguado, Ponz-Segrelles, Miura, Oguchi, Omori & Kohtsuka.
Ramisyllis multicaudata. Seven specimens (RM 1–7, Online Resource 2), Darwin Harbour, Channel Island (type locality), 12°33.2′S, 130° 52.4E′, coll. and identified by Glasby, Aguado & Ponz-Segrelles.
Syllis ramosa. 1 specimen University Museum of the University of Tokyo (UMUTZ-Ann-Pc-95) Found in the “gastral cavity and adjacent parts” of Crateromorpha meyeri rugosa in Sagami Bay (around 180 m deep). Coll. by K. Aoki and identified by A. Izuka (1912).
Syllis cf. ramosa. 1 specimen from the National Museum of Nature and Science of Tokyo (NSMT-Pol S. 1568). Found in “a sponge” at Sagami Bay (around 35–50 m deep), collected and identified by M. Imajima in 2005.
The name refers to King Ghidorah, the three-headed and two-tailed monster enemy of Godzilla. Both characters were created by Tomoyuki Tanaka based on Japanese mythology and folklore. King Ghidorah is a branching fictitious animal that can regenerate its lost ends. King Ghidorah is assumed to be a male and latinized accordingly.
Distribution and habitat
Coastal waters of Sado Island, Japan, around 15 m deep; symbiont of Petrosia sp. (pink form).
The sponges were collected on vertical stone walls, slopes, or small caves, usually in less exposed areas where they were often accompanied by other sponges, encrusting algae, and coralline algae. The sponges measured 5–10 cm in diameter and were usually irregularly round and pink, with mostly smooth surfaces except some areas showing crests, dead and healed areas (Fig. 5c), and some large oscula (Fig. 5d). Immediately after placing the sponges in trays, many very active, fast-swimming male stolons (see description below) left them. After 2–3 h, swimming female stolons (see description below) also left, moving slower than males. Detached stolons, mostly males, shook vigorously (Video S1), as in other syllids (MTA, personal observation). Dissection revealed only one worm specimen per sponge, most of them developing stolons (ten were males, five females), though not all of them showed signs of stolonization (four specimens). In sponges containing sexually mature specimens with attached stolons, some fully developed, detached stolons were also found in the sponge canals. All attached and free stolons from the same sponge specimen were of the same sex.
The anterior worm end, considerably less active than the posterior ends, was always at the inner basal area of the sponge. No pattern was observed in the position or orientation of the branches. The sponges were generally widely occupied, particularly in some areas. Worm branches were quite flexible and elastic, which facilitated fluent movement within the canal system. However, even though some branches could move outside the sponge when needed, worms were not able to abandon the sponge, even when some of their branches were dying. In natural conditions, the posterior ends emerged from the ostia or the oscula only in one specimen. In the laboratory, posterior ends moved on the sponge surface (Fig. 5e, f).
Dendriform branching body with one anterior and multiple posterior ends (Fig. 5a, b). Random branching asymmetry (Fig. 7h). Body subcylindrical, ventrally flattened, mostly translucent (except some yellowish or brownish areas in vivo). Holotype 0.36 mm wide at proventricle level, without parapodia. Branches always dichotomous, emerging at approximately right angles from intersegmental areas (Fig. 9a–d). Paired branches from same segment not seen. Holotype with first branching point after segment 24, second 4 and 6 segments later on each respective branch (Fig. 5a). Number of segments between two contiguous ramifications lacking obvious branching pattern (4–10 segments in holotype anterior branches to 10–20 segments between branching points in other regions). Most midbody segments as long as wide (70 µm length) (Fig. 9a–c, e–f), with some areas with much longer segments, 2–3 times as long as regular ones (174 µm; Fig. 9d), rectangular, yellowish or brownish, with much shorter dorsal cirri (Fig. 9d).
Prostomium rounded, with two pairs of eyes, anterior pair larger than posterior one; antennae articulated, median one slightly longer (8 articles in holotype) than lateral ones (6–7 articles in holotype) (Figs. 6a–c and 7a, c–d). Median antenna placed behind lateral ones (Fig. 6b). Palps small, conical, ventrally directed (Fig. 6b). Nuchal organs absent (Fig. 6b, c). Tentacular cirri articulated, dorsal ones longer (11 articles in holotype) than ventral ones (7 articles in holotype) (Fig. 6b, c). Dorsal surface of segments anterior to proventricle with a transversal band of cilia (Fig. 6c), then with bunches of cilia in proventricular segments (Fig. 10i) and with minute crests on midbody segments in one specimen (Fig. 10e–g).
Dorsal cirri usually straight, stretched horizontally in life (Fig. 7h), articulated, with those in segments anterior to first branching point longer (23 articles in holotype) than remaining ones (11–15 articles in holotype) (Fig. 5a). Anterior dorsal cirri as: 1st long, 2nd short, 3rd short, 4th long, 5th short, 6th long, 7th short, 8th short, and 9th long; remaining dorsal cirri generally with a strong long-short alternation in length (6–11 vs. 3–7 articles) (Fig. 9e, f). Longer dorsal cirri wider than short ones (Fig. 8). Some midbody branches with long yellowish segments (see above) and dorsal cirri with 1–4 articles lacking clear length alternation (Fig. 9d). Dorsal cirri length and shape symmetrical on each segment, but symmetry occasionally lost (one long and one short dorsal cirrus on same segment) (Fig. 7g). Dorsal cirri with spiral glands, larger and more remarkable in long ones (Figs. 8a, c-e, and 11a, c-f), opening exteriorly through both large and minute pores (joined in perforate plates) (Fig. 6d-f). Glandular content bright white in vivo, especially evident in long dorsal cirri (Fig. 7g), turning into intense red and massively protruding outside through pores when dying (Figs. 6g and 11b, f).
Ventral cirri short, unarticulated, digitiform to oval, basally inserted on parapodia, shorter than parapodial lobes, with numerous pores (Fig. 12g). Neuropodia bearing 2–3 simple chaetae (Fig. 12d-f), occasionally one (Fig. 12h), and one pointed acicula (Fig. 11g). Chaetae tomahawk shaped, bifid distally, prominent subdistal spur and series of denticles between teeth and spur; angle and relative sizes of distal teeth varies slightly along the body (Fig. 12a-f, h).
Pygidial cirri articulated, resembling dorsal cirri of posterior segments (9–10 articles) (Figs. 8e, f, and 10b). Numerous posterior ends regenerating with shorter dorsal and pygidial cirri (Fig. 10a). Anal openings densely ciliated (Fig. 10c, d).
Alimentary canal visible by transparency (Fig. 8a, c, i). Pharynx slender, through 12 segments in holotype, about one-fourth width of proventricle (Figs. 5a, 7a). Long, slender, cylindrical, strongly-cuticularized, with no tooth or trepan, partially eversible (Figs. 7d–f). Pharynx mostly straight, with a curve anterior to proventricle visible when moving (Fig. 7a). Proventricle prominent, barrel-shaped, almost as wide as body width, filling coelomic cavity, extending through 4–5 segments (15–18 in holotype) (Figs. 5a and 7a). Alimentary canal continuous through all branches. Content visible by transparency as a transparent fluid (Fig. 8a), occasionally with some brownish particles (Fig. 8c). No sponge tissue identified inside. Content of digestive tube moving through peristalsis in vivo, with posterior ends (last 10–20 segments, including anus) internally densely covered by cilia (Video S2) visible by transparency and through anus (Fig. 10c, d). A pair of nephridia per segment at basis of parapodia (Video S3). Each branch with a wide ventral blood vessel visible by transparency (Fig. 8h), ventral to, and wider than digestive tube (Fig. 8i), with a transparent fluid circulating inside and showing peristalsis (Video S4). Incomplete intersegmental anterior and posterior septa delimitate each segment. Digestive tube and ventral blood vessel slightly thinner when going through intersegmental septa (Fig. 8h). Nerve cord ventral, with multiple ramifications (Fig. 13a). Body wall muscles longitudinal, circular ones not seen. External body bifurcation at branching points accompanied by bifurcation of all longitudinal organs (ventral nerve cord, longitudinal muscles, digestive tube, and ventral blood vessel) (Figs. 13a, b), which occupy same relative position in new branches. “Muscular bridge” crossing dorsally over intestine and between ventral nerve cord and ventral blood vessel ventrally to one of three segments coming out from branching point (Fig. 13a, b), being delimitated by three Y-shaped intersegmental septa (Fig. 11h, i; Video S3).
Reproduction and regeneration
Sexes separate. Reproduction by gemmiparous schizogamy. Numerous stolons of same sex at end of terminal branches. Attached and detached stolons in a given host sponge are consistently of single sex (either male or female). Stalks undistinguishable from internodes (areas between two branching points) and other terminal branches lacking signs of gametogenesis (Fig. 14i). Segments from stalk with clear alternation in dorsal cirri length (Fig. 14i). No correlation between number of stalk segments and stolon maturity. Ventral regeneration of stalk pygidium starting before stolon detachment (Figs. 13h and 14g, h). Stalks with recently detached stolons showing stubby endings, still ventrally directed, with signs of stolon attachment dorsally, clearly differing from growing tips of new stolons or of developing branches (Figs. 8b, g, and 13f). When dorsal surfaces are repaired, a pair of anal cirri and a new anal opening are developed, followed by regular growth and addition of segments just in front of newly formed pygidium (Fig. 12).
Stolons acerous, with bilobed anterior end, lacking antennae and palps (Figs. 13c, d, and 14a–f). Two pairs of well-developed dorsal (posterior) and ventral (anterior) eyes; ventral pair larger than dorsal one (Fig. 14g, h). A vestigial digestive tube through males and female’s stolon segments, bubble like in female first segments, very narrow in remaining segments. Mature female and male stolons having dense bundles of long paddle-like natatory chaetae in addition to typical stock neurochaetae, transparent, long, distally pointed (“wing- or leaf-like”) (Fig. 13e), developing in mature stolons, usually seen in recently detached ones. Not seen in still attached, non-fully developed stolons.
Male stolons similar in size to female stolons, but with considerably longer parapodia and narrower bodies (Figs. 13c and 14f), with first two pairs of dorsal cirri longer than following ones (Fig. 14f) and internal oval structures at basis of parapodia (Fig. 13i) (possibly chaetal sacs of the paddle-like chaetae), with first three segments full of yellowish sperm (regionalization) (Fig. 14f). Female stolons with marked positive phototaxis when mature and detached (Video S5), all dorsal cirri of about same length, and segments full of oocytes, even in parapodia (Fig. 14a–e), pink in detached females, white in not completely developed females (Fig. 14c–e). Developing larvae or embryos not observed.
Ramisyllis kingghidorahi n. sp. and R. multicaudata (Glasby et al., 2012; Ponz-Segrelles et al., 2021; Schroeder et al., 2017) differ from S. ramosa (except the Red Sea and Imajima’s 2005 Sagami Bay specimens) in living from 0 to 20-m depth inside species of Petrosia instead of 100–1000 m depth inside species of Crateromorpha (Izuka, 1912; McIntosh, 1879; Oka, 1895); in having the proliferating area after the parapodia and never replacing it or the dorsal cirri (Glasby et al., 2012) instead of new branches emerging from the parapodium and lacking dorsal cirri as in S. ramosa; in lacking two branches emerging from both sides of the same segment, which may occur in S. ramosa (Pl. XXIII, Fig. 11 in McIntosh,; Fig. 2 in Oka, 1895); in having simple, robust, tomahawk-shaped chaetae instead of slender, hooked at the tip and a fusion line between shaft and blade in S. ramosa (Pl. XVIA; Fig. 1 in McIntosh, 1885); and in newly formed branches acquiring very soon the segment size and cirri length of previous branches instead of showing differences in segments width and smaller and shorter than usual dorsal cirri in S. ramosa (e.g., Fig. 18 in Okada (1937)).
Ramisyllis kingghidorahi n. sp. lives inside an undescribed Petrosia sponge and R. multicaudata in another unidentified species of Petrosia (probably Petrosia cf. nigricans, pers. comm. Dirk Erpenbeck), in both clearly different ecosystems (costal coral reef vs. rubble sand with algae, respectively) at different latitudes with different water temperatures. Dorsal cirri are generally longer in R. kingghidorahi n. sp. than in R. multicaudata, particularly in the anterior end (Fig. 7a, b; Online Resource 11), the proventricle is also longer (4–5 vs. 2–4 segments) (Online Resource 11), stalks are similar to segments in regular branches, while these are narrower with shorter dorsal cirri in R. multicaudata (as in S. ramosa) and their stolons also slightly differ in the relative length of some features (e.g., dorsal and ventral cirri, Online Resource 12). In R. kingghidorahi n. sp. the development of a new branch seems to occur just in the intersegmental area, while it was described that in R. multicaudata, it begins in front of the posterior septum (Glasby et al., 2012). In R. kingghidorahi n. sp. intersegmental septa of the two pre-existing segments and the newly formed one can be observed by transparency (Fig. 11h, i); they form a “Y” shape (Fig. 11h, i,; Video S3), and appeared to be reduced as in R. multicaudata (Ponz-Segrelles et al., 2021), allowing the thinner digestive tube and ventral blood vessel to pass through them (Fig. 8h, i). Nevertheless, in both species, all longitudinal organs bifurcate in the branching points, new branches show internal muscular bridges crossing between the different organs and the ventral blood vessel is considerably enlarged in comparison with other syllids and similar in diameter to the digestive tube (Ponz-Segrelles et al., 2021).
The three branching syllids show segmental asymmetry (i.e., segments with pairs of dorsal cirri of different length on each side), which intervenes between regions of symmetry (Schroeder et al., 2017) and have been found to show reddish coloration (Glasby et al., 2012; Imajima, 1966; Read, 2001). The glandular material of dorsal cirri in R. multicaudata changes from bright white into red colour when the animals start dying (Ponz-Segrelles et al., 2021), and we observed a similar phenomenon in R. kingghidorahi n. sp., with this material protruding through the dorsal cirri pores.
The precise behaviour of female stolons once detached was not determined in R. multicaudata, although the presence of paddle chaetae suggested enhanced swimming ability (Ponz-Segrelles et al., 2021; Schroeder et al., 2017). In R. kingghidorahi n. sp., female stolons showed a clear positive phototaxis (Video S5) which, together with the paddle-like natatory chaetae, suggests that they leave the sponges for spawning. Syllis ramosa from the Philippines type locality might be viviparous (McIntosh, 1885), but this was neither confirmed for the type material by Glasby et al. (2012), nor for the two species of Ramisyllis.
Previous reports of S. ramosa could represent more than one branching species (Glasby et al., 2012), including, for instance, the Red Sea specimen inhabiting a shallow water silicious sponge. Indeed, in agreement with Leslie Harris (pers. comm.), we suggest that a picture by Danièle Heitz of a Petrosia from the Red Sea (Al Birk) with syllid branches emerging from one osculum (https://nomadica.jimdofree.com/vers-marins/annélides/ramisyllis-multicaudata/) likely corresponds to an undescribed species of Ramisyllis. The specimen from Sagami Bay identified by Imajima in 2005 shows differences in chaetal morphology, compared with the three currently known branching species and, thus, might also be an undescribed species. The report of S. ramosa from the southern coast of Jeju Island in South Korea (Lee, 1992) is herein considered as dubious, since it was found on mollusc shells and had compound chaetae.