Typosyllis heronislandensis Hartmann-Schröder, 1991

Aguado, María Teresa, Helm, Conrad, Weidhase, Michael & Bleidorn, Christoph, 2015, Description of a new syllid species as a model for evolutionary research of reproduction and regeneration in annelids, Organisms Diversity & Evolution (New York, N. Y.) 15 (1), pp. 1-21 : 6-15

Typosyllis heronislandensis Hartmann-Schröder, 1991 . Two paratypes ZMH P-20539.

Description

External anatomy

Holotype with 70 segments and one of the longest complete paratypes with 85 segments, no reproductive signs, 15 mm long, 0.8 mm wide. Before reproduction, specimens can reach up to 80–120 segments. The body is long, cylindrical in section, and ventrally flattened. Dorsal color pattern consists of transversal dark brown lines on dorsum of anterior segments ( Fig. 1a–c ), one line per segment. Posterior segments have no pigmentation, and digestive tube is visible by transparency ( Fig. 1a ). The first segment has one curved spot, medially located on the dorsum and strongly pigmented in dark red or brown ( Fig. 1a, b ). Color pattern is more distinct in the region anterior to proventricle ( Fig. 1a ). In addition to dark brown transversal lines, proventricle segments occasionally with some dark brown spots on the dorsum that are irregularly distributed. Ethanol-preserved specimens are yellowish and with no pigmentation. Prostomium is wide and long, with two pairs of eyes in trapezoidal arrangement, anterior ones slightly larger than posterior pair, eye spots are absent. Antennae, tentacular, and dorsal cirri are long ( Fig. 2a, b, d ). Median antenna is inserted on the middle of prostomium, with ca. 30 articles; lateral antennae are shorter, inserted on the anterior margin of prostomium, with ca. 20 articles ( Fig. 1a ). Palps are triangular, longer than prostomium ( Fig. 2c ), and fused at base with a distinct median groove. Nuchal organs form two ciliary grooves between prostomium and the first segment. The first segment is similar in length to subsequent segments. Two pairs of tentacular cirri are present; dorsal tentacular cirri are longer than the antennae ( Fig. 2a ), with ca. 40 articles, ventral ones are shorter than the dorsal ones, with ca. 30 articles. Dorsal cirri are alterning in orientation and length. Long dorsal cirri have ca. 35 articles, and short ones have ca. 20 articles in anterior segments ( Fig. 2a ); in midbody and posterior segments, long dorsal cirri have ca. 30 articles and short ones ca. 20 articles ( Fig. 2e ). Longer ones are pointing up and shorter ones pointing down. Spiral glands are inside articles. Ventral cirri digitiform are inserted proximally and reaching tips of parapodia ( Fig. 2b, f ). Conical parapodia each have prechaetal and postchaetal lobes, whereas the latter is more distinct ( Fig. 2f ); lobes are larger in anterior and midbody segments. All chaetae compound and falciger-like, bidentate with distal and proximal teeth similar in length. Spinulation on edge is long, reaching the proximal tooth but not extending beyond it. Anterior parapodia have seven to ten compound chaetae, bidentate blades, gradating in length (most dorsal ca. 28 μm, most ventral ca. 14 μm), and blade edge has long spines through the whole edge ( Figs. 3a–c and 4a–c ); most distal spines reach the level of the proximal tooth ( Fig. 4b ). Midbody parapodia have six to seven compound chaetae, blades bidentate, similar in length to anterior ones, with long spines through the whole edge ( Fig. 3d–f ); most distal spines reach the level of the proximal tooth ( Fig. 4e, f ). Posterior chaetigers have four to six compound chaetae, blades bidentate, shorter than those of anterior chaetigers (most dorsal ca. 24 μm, most ventral ca. 10 μm) and have long spines through the whole edge ( Fig. 3g, j ); most distal spines reach the level of the proximal tooth ( Fig. 4j–m ). Most ventral chaetae are in posterior segments with pointed shafts ( Figs. 4l and m ). Posterior segments have one bidentate ventral simple chaeta ( Figs. 3h and 4h ) and one dorsal simple chaeta with a distal notch ( Figs. 3h, i and 4g ). Ventral and dorsal simple chaetae have small spines on distal end ( Figs. 3h, i and 4h ). Anterior parapodia have three to four pointed aciculae, one to two slightly distally bent ( Fig. 4d ); midbody parapodia have two to three pointed aciculae and one thin acicula in addition. Occasionally, one to two aciculae are protruding from parapodia. Most posterior segments have one pointed acicula and one additional thin acicula ( Fig. 4i ). Pygidium conical, with two anal or pygidial cirri (ca. 30 articles) and one median papilla ( Fig. 3l ) present in most of the specimens. Articles of anal cirri have pores ( Fig. 3k ).

Internal anatomy

The musculature of the body wall is characterized by two major muscle systems, the outer transversal arranged muscle fibers (often named circular muscle fibers in other annelids) and the inner longitudinal muscles ( Figs. 5b–d and 6a ). Whereas the longitudinal muscle fibers form distinct bundles, the transverse fibers form a dense meshwork of fibers partly encircling most parts of the dorsal and ventral body ( Fig. 5b, c ). Notably, the transverse fibers do not form a prominent layer embracing the whole animal but exhibit a gap of fibers at the ventral side and therefore cannot be named circular ( Fig. 6b ). Furthermore, most dorsal fibers being part of the outer muscle layer are not restricted to only one parapodial region. These fibers interconnect two parapodial regions and as a consequence, represent a diagonal cross bracing pattern. The latter pattern is most prominent in anterior dorsal regions ( Fig. 5c ) but is lacking in posterior body parts.

Whereas the transverse fibers form a consistent outer layer, the longitudinal fibers are arranged in prominent bundles. Thus, two prominent longitudinal muscle bundles are situated each at the dorsal and ventral side ( Figs. 5b–d and 6a–c ). Notably, the dorsal longitudinal bundles are represented by two distinct muscular plates ( Fig. 6a ). The anterior origin of the dorsal bundles can be found in the antennal region, whereas the ventral bundles originate anteriorly to the first chaetiger ( Fig. 5b ). The pygidium lacks distinct longitudinal and transverse muscle bundles ( Fig. 5d ).

Palp muscles insert in dorsal anterior regions, anterior of the antennal base ( Fig. 5a, b ). The musculature is represented by a prominent median muscle fiber and weaker muscle fibers branching off the main muscle. Notably, the margin of the palps lacks muscle fibers. The antennae and dorsal and ventral cirri are quite similar in terms of muscle arrangement (see Fig. 5a–f ). Each antenna and cirrus is formed by inner longitudinal muscle fibers running from the base to the apex ( Fig. 5a–c, e ). In the dorsal cirri, additional lateral F-actin staining is visible, exhibiting a striped pattern ( Fig. 5f ). The base of the dorsal cirri is formed by distinct cirral muscle bundles ( Fig. 6a ) possessing a basal muscular socket. The base of the antennae is represented by a muscular socket, too ( Fig. 5a ). In contrast to the dorsal cirri, the antennal base is formed by separate muscle fibers terminating in the region of the peristomium ( Fig. 6a ).

The uniramous parapodia insert laterally between the dorsal and ventral longitudinal muscle bundles ( Fig. 6a ). The parapodium itself consists of distinct parapodial retractor muscles forming the parapodial cone and prominent acicular protractor muscles inserting at the aciculae ( Fig. 6a ). The latter muscle fibers terminate at the aciculae and under the epidermis, in close proximity to the longitudinal muscle bundles. Furthermore, the acicular flexor muscle terminates at the acicular base and runs towards ventral where it is anchored between the ventral longitudinal muscle fibers ( Fig. 6a ). Another distinct muscle bundle is formed by the chaetal flexor muscle that runs from the apical chaetae toward ventral and terminates in the region of the median muscle bundle ( Fig. 6a ). Additionally, the base of the dorsal cirrus is formed by cirral muscle fibers that run from the parapodial cone toward the cirral base ( Fig. 6a ).

Dorsal and ventral simple chaetae, posterior parapodium. i Dorsal simple chaeta, posterior parapodium. j Chaetal fascicle, posterior parapodium. k Anal cirri, detail of pores. l Pygidium and anal cirri, ventral view

The internal pharynx is similar in length to the prominent proventricle. Thus, the whole structure is visible through 13– 15 segments, and the proventricle itself extends through eight segments ( Fig. 1c ), with about ca. 30 rows of muscular cells, visible by transparency and cLSM ( Fig. 6d, e ). Furthermore, a tooth on the anterior margin ( Fig. 1a, b ) and ten distal papillae are visible ( Fig. 5a, b ). The pharynx musculature is represented by a dense meshwork of longitudinal and circular muscle fibers, which form a tube ( Fig. 5a ). The proventricle almost fills out the entire body cavity ( Fig. 6b ) of the anterior animal. Forming a muscular tube, the proventricle has a slitlike passage where the intestine is located ( Fig. 6b ). The staining against serotonin reveals a strong homogenous serotonergic immunoreactivity within the whole proventricle posterior parapodium. i Posterior aciculae. j Posterior chaetae, most dorsal ones and medially located in the fascicle. k Posterior chaetae, most dorsal ones. l, m Posterior chaetae, most ventral ones. Arrows pointing the distal spines that reach the level of the proximal tooth and pointed posterior shafts (l, m)

( Fig. 6c ). The muscular tube itself is formed by honeycomb-like radial muscle bundles in ca. 30 rows, which are enveloped by distinct circular muscle fibers ( Fig. 6d ). At the anterior end of the proventricle, prominent suspending muscles are exhibited, anchoring the proventricle within the body cavity. Furthermore, the anterior end of the proventricle is characterized by a tube-like anterior circular muscle layer ( Fig. 6d ). After the proventricle, there is the ventricle, followed by two caeca. Methylene blue staining reveals spherical structures within the caeca strongly pigmented in blue.

Reproduction

Long specimens (usually with 80–120 segments) develop stolons. Stolonization seems to occur once a month under laboratory conditions (when temperature is more than 24 °C, circadian rhythm with 12 h light). Adults produce gametes in their midbody and posterior segments (approximately from segments after the proventricle until the pygidium). Small disturbances provoke the animal to release gametes laterally, possibly through the nephridia ( Fig. 7g ). When the stolon is formed and still attached to the parental body, gametes move toward the posterior segments ( Fig. 7d, f ). Stolons are dicerous, with two anterior lobes, two pairs of conspicuous eyes and two small antennae anteriorly located ( Fig. 7e ). Female stolons are full of spherical oocytes gray in color ( Fig. 7b, d ) and male stolons with two yellowish packages of sperm per segment ( Fig. 7a, c ). Typosyllis antoni n. sp. shows sequential hermaphroditism.

Ecology

Animals live at the bottom, often within the soft sediments. They feed on algae. All the specimens have been found with some algae material in their digestive tubes.

Regeneration

T. antoni n. sp. regenerates anteriorly and posteriorly. After dissection, the wound site constricts by musculature, forming an invagination of the first remaining segment. Two days after dissection, blastema formation occurs in anterior regenerating specimens, whereas this process is delayed in posterior regeneration. Both anterior and posterior regenerating specimens bear a well-developed blastema after 3 days. Four days after dissection, the regenerated anterior end bears a prostomium with developing antenna, palps, and eyes ( Fig. 8a ). Additionally, new segments become visible, with regenerating tentacular cirri in the first segment. The number of re-established anterior segments varies between two and three in different specimens but is limited to the initially existing number. At the same stage, the posteriorly regenerating specimens exhibit a pygidium with developing anal cirri and a median papilla ( Fig. 8b ). During the following days, the anterior structures grow and 6 days after dissection, a pharynx is visible ( Fig. 8c ), connecting the intestine with the mouth opening. In posterior regenerating specimens, the process is continued by addition of segments, first visible at day 5. After 6 days, posterior segments bear cirri and chaetae become visible ( Fig. 8d ). Moreover, the anal cirri have developed the first articles. The second week is dominated by growth in both cases of regeneration. Regenerated anterior and posterior ends are still small, but the difference to the residual body segments becomes indistinct. Remarkably, there are no signs of a regenerated proventricle, ventricle, caeca, or pharyngeal tooth in the anterior part ( Fig. 8e ). In contrast, the posterior regenerating specimens reach the original adult shape ( Fig. 8f ). Many of the anterior regenerating specimens develop gametes and stolons. In both plastic bowls as well as in the control, specimens with regenerating posterior ends and complete untouched ones show signs of stolonization. Free stolons were also observed during the experiment. Further experiments show evidence of regeneration in more than two anterior chaetigers when individuals are dissected directly in front of the proventricle. Moreover, when dissecting between chaetigers 35 and 36 or when dissecting directly behind the proventricle, decapitated specimens produced two or three sequential stolons in a few cases. In one case, the regeneration of the posterior end occurred, while the stolon was still attached. Finally, regeneration of posterior segments after stolonization appears to be delayed, when specimens where dissected between chaetigers 35 and 36.

Remarks

Considering external morphological features, T. antoni n. sp. is different to all other species of the Syllis - Typosyllis group in the following combination of characters: color pattern consisting in transversal red lines on the dorsum of anterior segments; antennae and dorsal cirri long, the latter with strong intestine (in). d1, d2 The posterior end is well regenerated after 6 days, with five segments (I–V). The older segments have re-developed their cirri (dc, vc) and the chaetae (arrows). e The regeneration of the anterior end is at an advanced stage after 14 days. Any signs of proventricle, ventricle, or ceca or pharyngeal tooth are missing. f At 14 days, the regeneration of the posterior end has nearly finished. 1–3, regenerated anterior segments; I–VIII, regenerated posterior segments; ac anal cirrus, as anus, dc dorsal cirrus, dt dorsal tentacular cirrus, ey eye, in intestine, la lateral antenna, ma median antenna, mp median papilla, ph pharynx, pl palp, ps prostomium, py pygidium, vc ventral cirrus, vt ventral tentacular cirrus. Scale bar =100 μm

alternation in length; bidentate chaetae falciger-like with proximal and distal teeth similar in length and long spinulation on edge; one tiny and thin acicula appearing in posterior segments in addition to thicker and pointed one and a long proventricle.

T. antoni n. sp. is most closely related to T. heronislandensis Hartmann-Schröder 1991 (from East Australia). It is similar to this species and Syllis parturiens Haswell 1920 (Gulf of Aqaba and Australia) (not included herein) in chaetae shape ( Hartmann-Schröder 1991; Haswell 1920). However, T. heronislandensis and Typosyllis parturiens have different spinulation; long spines are restrict- ed to the base of anterior blades, while they become much shorter through the distal end ( Hartmann-Schröder 1991; Licher 1999). In T. antoni n. sp., the spinulation is long through the whole edge in all the chaetiger blades from anterior and posterior segments. In all blades, most distal spines remain long and reach the level of proximal tooth (see arrows in Fig. 4 ). Additionally, T. heronislandensis has short dorsal cirri, and S. parturiens is viviparous ( Licher 1999). Moreover, T. heronislandensis seem to lack a distinctive color pattern ( Hartmann-Schröder 1991), although this is usually not a good diagnostic character since specimens frequently loose coloration when they are preserved. Finally, none of these two species have an additional thin acicula in the posterior segments. Typosyllis horrockensis Hartmann-Schröder 1981 (from West Australia) is a similar species in the shape of blades and color pattern of live specimens, but it has short dorsal cirri and short spinulation of blades ( Hartmann-Schröder 1981; Licher 1999). Typosyllis filidentata Hartmann-Schröder 1981 (Patagonia and West Australia) has chaetae with long spinulation ( Hartmann-Schröder 1979); however, shape of blades and length and orientation of spines is different to those found in T. antoni n. sp. T. antoni n. sp. is also characterized, among other features, by the curvature of the distal and proximal teeth of blades. Similar shape of blades’ teeth is found in Syllis augeneri Haswell 1920 ( Australia and Indonesia) and Typosyllis edensis Hartmann-Schröder, 1989 ( Australia) ( Hartmann-Schröder 1989; Haswell 1920; Licher 1999; Aguado et al. 2008). However, these species have shorter dorsal cirri and different aciculae.

As stated above, another distinct feature of T. antoni n. sp. is the presence of an additional considerably thin acicula in posterior segments. This kind of acicula has been described before for other species, such as Syllis westheidei ( San Martín 1984) (from Galapagos, Red Sea, and Mediterranean Sea) and Typosyllis magna ( Westheide 1974) (from Galapagos, and Gulf of Aqaba). However, none of these species share all the characteristics of T. antoni n. sp. S. westheidei has a different shape of blades and color pattern, and Syllis magna has pseudospiniger or spiniger-like blades.

Etymology

This species is dedicated to Anton Helm, who was born approximately when the first specimens were studied and identified as possibly new.

Phylogenetic results

The results of the different methodologies when analyzing the combined data set widely agree in the recovered topologies, excepting for the position of some few taxa ( Figs. 9 and 10 ). The main differences regard to the placement of Parahaplosyllis brevicirra Hartmann-Schröder 1991 , which is sister to Syllis alternata Moore, 1908 , in ML results ( Fig. 9 ), while sister to Trypanosyllis / Xenosyllis / Eurysyllis clade in MP (1 most parsimonious tree (MPT), 15,354 steps) ( Fig. 10 ); in both cases, support values are not high (B, 72 %; JK, 56 %). Another difference when comparing both topologies ( Figs. 9 and 10 ) is the position of clade 2, but again, support values of its sister relationships are, in both cases, considerably low.

Results show the monophyly of Syllinae well supported, mainly in the ML result (B, 94) ( Fig. 9 ). The sister group of Syllinae is the monophyletic genus Perkinsyllis Aguado and San Martín 2009 . Syllinae is divided into different clades, some of them with high support values, such as the following: Trypanosyllis / Xenosyllis / Eurysyllis ; Haplosyllis Langerhans, 1879 ; Branchiosyllis Ehlers, 1887 ; Paraopisthosyllis Hartmann-Schröder, 1991 ; Megasyllis San Martín, Hutchings, and Aguado, 2008 ; clade 1 and clade 2. The genera Typosyllis , Syllis , and Opisthosyllis are not monophyletic ( Figs. 9 and 10 ).

A sister group relationship of T. antoni n. sp. and T. heronislandensis Hartmann-Schröder, 1991 , is supported by both methodologies (ML and MP) and by very high support values (B and JK, 100) ( Figs. 9 and 10 ). Syllis / Typosyllis species with spiniger-like chaetae are located within clade 1 (such as Typosyllis yallingupensis Heacox and Schroeder, 1982 , and Syllis garciai Campoy, 1982 ). Syllis / Typosyllis species with simple chaetae that appear after a fusion process of shafts and blades (such as Syllis gracilis Grube, 1840 ; Syllis ypsiloides Aguado, San Martín, and Ten Hove, 2008 , and S. cf. gracilis australiensis Hartmann-Schröder, 1979) are located in different clades.

Figures

Fig. 1 Light microscopy pictures of Typosyllis antoni n. sp. a Live specimen, anterior end, dorsal view. b Detail of anterior end, dorsal view. c Detail of proventricle, dorsal view. Scales a 0.8 mm; b, c 0.3 mm

Fig. 2 SEM images of Typosyllis antoni n. sp. a Anterior end, dorsal view. b Anterior end, ventral view. c Detail of anterior end, dorsal view. d Anterior end, lateral view. e Midbody segments, ventral view. f Midbody parapodia, lateral view

Fig. 3 SEM images of Typosyllis antoni n. sp. a Chaetae fascicle, anterior parapodium. b, c Most dorsal chaetae, anterior parapodium. d, e Most dorsal chaetae, posterior parapodium. f Medially located chaetae, posterior parapodium. g Most ventral chaetae, posterior parapodium. h

Fig. 4 Light microscopy pictures of Typosyllis antoni n. sp. a Anterior chaetae, most dorsal ones and medially located in the fascicle. b Anterior chaetae, most dorsal ones. c Anterior chaetae, medially and most ventral ones. d. Anterior aciculae. e Midbody chaetae, most dorsal ones and medially located in the fascicle. f Midbody chaeta, most dorsal one. g Dorsal simple chaeta, posterior parapodium. h Ventral simple chaeta,

Fig. 5 Confocal maximum projections of Typosyllis antoni n. sp. Phalloidin–rhodamine labeling of adult specimens. Anterior is up in b and c, down in a and d, and right in e and f. a Lateral view of the anterior region, with everted pharynx (ph) and prominent dorsal cirri (dc), antennae (an), and palps (pl). The antennae exhibit prominent basal muscular sockets (dotted ring). b Ventral view of the anterior region showing the distinct longitudinal muscle bundles (lm) and the transverse muscle fibers (tm) forming semicircular fibers. c Dorsal view of the anterior region. Distinct longitudinal muscle fibers (lm) and the partly diagonal cross bracing transverse muscle fibers (tm) are exhibited. d Dorsal view of the posterior end showing the prominent pygidium (py) lacking distinct muscle bundles and the pygidial cirri (pc). e Lateral view of the midbody region, dorsal is up. The dorsal cirri (dc) insert at the base of the parapodium (pa). f Dorsolateral view of a midbody parapodial region. The dorsal cirri (dc) are represented by a prominent staining exhibiting a striped pattern; an antenna, ch chaetae, dc dorsal cirrus, lm longitudinal muscle fiber, mo mouth opening, pa parapodium, pc pygidial cirri, ph pharynx, pl palps, pp pharyngeal papillae, pr parapodial retractor muscle, py pygidium, tm transverse muscle fiber, vc ventral cirrus.Scale bar= 100 μm

Fig. 6 Confocal maximum projections of Typosyllis antoni n. sp. Phalloidin–rhodamine (gray) and serotonin labeling (red) of cross sections (a–c) and the proventricle (d, e) of adult specimens. Dorsal is up in a–c. Anterior is up in d and right in e. a Cross section of the midbody region, the prominent longitudinal muscle bundles are colored in yellow. The insert shows a detailed view of the parapodium, and major muscle bundles are color coded— parapodial retractor muscle in brown, acicular protractor muscle in green, acicular flexor muscle in violet, chaetal flexor muscle in pink, cirral muscle bundle in blue. b Cross section showing the distinct proventricle (pr) filling almost the whole body cavity. The longitudinal muscle bundles are color coded in yellow. c The proventricle (pr) and the ventral nerve cord (vn) show distinct serotonergic immunoreactivity. d The separated proventricle (pr) exhibits radial honeycomb-like muscle bundles (rb, dotted circle) and prominent circular muscle fibers (cf) surrounding the whole structure. e The separated proventricle (pr) is represented by an anterior circular muscle bundle (am) and suspending muscles (sm) terminating at the border between the anterior circular muscle bundle (am) and the radial muscle bundles (rb). am anterior circular muscle bundle, cf circular muscle fiber, dc dorsal cirrus, dv dorsoventral muscle fibers, in intestine, mm median muscle bundle, pa parapodium, pr proventricle, rb radial muscle bundle, sm suspending muscle fiber, vn ventral nerve cord. Scale bar=100 μm (color figure online)

Fig. 7 Typosyllis antoni n. sp. a Male stolon, dorsal view. b Female stolon, dorsal view. c Male stolon still attached to the parental body, dorsal view. d Female stolon still attached to the parental body, dorsal view. e Detail of anterior appendages of male stolon, dicerous kind, dorsal view. f Ovaries within posterior segments of parental body and female stolon still attached. g Posterior segments with ovaries of a female that has not developed a stolon yet

Fig. 8 Light microscopy pictures of regenerating specimens of Typosyllis antoni n. sp. All pictures are dorsal views except d2 (ventral view). a, c, e anterior end, b, d1, d2, f posterior end. The dotted white line indicates the site of dissection. a At 4 days after dissection, the re-developing prostomium (ps), as well as the first two segments (1, 2) are visible (in other specimens also three segments were observed). The prostomium shows palps (pl), antenna (la, ma) and two pairs of eyes (ey). Dorsal tentacular cirri (dt) of the first segment occur. b After 4 days, the pygidium (py) with the anal cirri (ac) and the median papillae (mp) are regenerated. c At 6 days, all in a described anterior structures have grown. Pharynx (ph) is re-developed, connecting the mouth opening (not visible) with the

Fig. 9 Maximum likelihood tree. Bootstrap support values above nodes. Syllis and Typosyllis species as they were described. Drawing: S. marugani (after Aguado et al. 2006)

Fig. 10 Most parsimonius tree. Jacknife support values above nodes. Syllis and Typosyllis species as they were described. Drawings from up to down: chaetae of Syllis benbeliahue (after Aguado and San Martín 2006),