Polydora tunicola Abe, Hoshino & Yamada, 2022

Abe, Hirokazu, Hoshino, Osamu, Yamada, Kazuyuki, Ogino, Tetsuya, Kawaida, Shun & Sato-Okoshi, Waka, 2022, A novel symbiotic relationship between ascidians and a new tunic-boring polychaete (Annelida: Spionidae: Polydora), Zootaxa 5159 (1), pp. 1-22 : 7-15

Polydora tunicola Abe, Hoshino & Yamada , sp. nov.

[Japanese name: Hoyano-poridora]

( Figs. 2–4 )

urn:lsid:zoobank.org:act:45399271-214B-42C0-9DAF-40661AEE1B2C

Spionidae sp.: Hoshino 2016: page 50–51, 3 figs.

Polydora sp. : Hoshino 2020: page 100–105, 9 figs.

Type material. Holotype: NSMT-Pol H-855, Akinohama , 34.7868 N, 139.4088 E, Izu-Oshima Island, Tokyo Pref., subtidal, 10 m depth, rocky shore, isolated from Polycarpa cf. cryptocarpa kroboja, 04 August, 2016 ; 35 Paratypes: NSMT-Pol P-856, Akinohama, 34.7868 N, 139.4088 E, Izu-Oshima Island, Tokyo Pref., subtidal, 10 m depth, rocky shore, isolated from Polycarpa cf. cryptocarpa kroboja, 04 August, 2016; 1 Paratype: NSMT-Pol P-857, Sakai Port, 33.7436 N, 135.3325 E, Minabe Town, Wakayama Pref., isolated from Polycarpa sp. , 01 April, 2021.

Holotype description. A complete 107-chaetiger individual, measuring 27 mm long and 1.1 mm wide at chaetiger 5. Body light tan in alcohol ( Fig. 2 ) and yellow in life ( Fig. 3A–C ); yellow pigmentation present on prostomium, peristomium, dorsal, ventral, and lateral sides of body in preserved specimen ( Fig. 2 ), but difficult to observe in fresh condition ( Fig. 3A–C ). Prostomium rounded on anterior margin ( Figs. 2B , 3A ); eyes absent; caruncle extends posteriorly to middle of chaetiger 3, with small median antenna present on caruncle at level of chaetiger 1. Palps extending posteriorly for about 11 chaetigers, color in alcohol opaque white to light tan, without black pigmentation.

Chaetiger 1 with small noto- and neuropodial postchaetal lamellae compared to subsequent chaetigers ( Fig. 3A ); notochaetae absent, short capillary neurochaetae present. Postchaetal lamellae well developed on anterior chaetigers except chaetiger 5, gradually reduced on posterior chaetigers. Prechaetal lamellae absent in all parapodia. Neuro- and notochaetae of chaetiger 2–4, 6 winged capillaries, arranged in two rows; capillaries in anterior row more curved and winged than those in posterior row. Number of capillaries per fascicle and wings in capillaries in notopodia of succeeding chaetigers diminished gradually; rows of notochaetae becoming indistinct in posterior chaetigers. Posterior notochaetae only capillaries. Hooded hooks of neuropodia from chaetiger 7 ( Fig. 3B, C ), not accompanied by capillaries; hooks bidentate, with main fang at right angle to shaft and acute angle with apical tooth; slightly curved shaft having constriction in upper part; curve and constriction of shaft weak in hooks from 7th chaetiger ( Fig. 3G ) but develop gradually toward posterior chaetigers ( Fig. 3H, I )); hooks up to 12 in series.

Chaetiger 5 slightly larger than either chaetiger 4 or 6; not overlapping chaetiger 6 ( Figs. 2B , 3A ); with curved oblique row of 10 dorsal falcate major spines alternating with pennoned companion chaetae ( Fig. 3F ); Anterior spines in a row falcate with lateral tooth; posterior spines in a row simple falcate or falcate with a rough-edged transverse shelf ( Fig. 3F ). Ventral tuft of four short winged capillaries present ( Fig. 3C ).

Branchiae from chaetiger 7 to end of body, absent only from six posterior-most chaetigers, short, not overlapping mid-dorsal, full-sized from chaetiger 9 or 10 to middle of body, then decreased in size; free from notopodial postchaetal lamellae, with flattened surfaces oriented parallel to body axis.

Pygidium wide disc with mid-dorsal gap; white-colored with black rim ( Fig. 2D–F ). No gizzard-like structure in digestive tract. Glandular pouches in neuropodia from chaetiger 7 onward, largest in chaetigers 9–12 ( Fig. 3D ).

Variability. The largest individual 27 mm long, 1.1 mm wide for 107 chaetigers (Holotype). Body pigmentation brown and/or yellow may be present on palp, prostomium, peristomium, dorsal and ventral sides, and pygidium. Bands of black pigment, probably remnants of larval pigmentation, on dorsal side of anterior chaetigers in small individuals. Prostomium rounded or weakly incised on anterior margin. Eyes usually absent in large worms, but occasionally present in small individuals especially in juveniles; four black eyes arranged as trapezoid shape, anterior pair widely spaced, posterior pair closely spaced. Caruncle extending posteriorly to end of chaetiger 2 or 3. Median antenna fingerlike ( Fig. 3A ) or small and indistinct ( Fig. 2B ). Palp length varied depending on degree of extension at fixation, extending as long as body length in live individuals especially in juvenile. Tip of anterior most spines in a row in chaetiger 5 occasionally chipped. Tip of companion chaetae often feather-like especially anterior in a row ( Fig. 3F ). Chaetiger 5 without dorsal superior capillaries but with ventral capillaries. Pygidium cup-shape to a wide flare-disc with mid-dorsal gap; white colored with black or brown rim. Pygidium of several specimens without black or brown rim, possibly due to intraspecific variation, but probably also due to susceptibility to tearing at this region.

Remarks. Polydora tunicola sp. nov. is similar to P. aura Sato-Okoshi, 1998 , P. cornuta Bosc, 1802 , P. fusca Radashevsky & Hsieh, 2000 , P. glycymerica Radashevsky, 1993 , P. latispinosa Blake & Kudenov, 1978 , P. lingulicola Abe & Sato-Okoshi, 2020 , P. nanomon Orensky & Williams, 2009 , P. robi Williams, 2000 , and P. vulgaris Mohammad, 1972 in having a median antenna on the caruncle and chaetiger 5 without dorsal superior capillaries, but with ventral capillaries. Polydora tunicola sp. nov. can be distinguished from these similar species by combining morphological characteristics such as maximal caruncle length, number of branchial chaetigers, absence of modified posterior notochaetae, yellow body pigmentation, and unique black-rimmed flaring pygidium ( Table 2 ). Polydora tunicola sp. nov. closely resembles P. cornuta in both having caruncle continued to the end of chaetiger 3, branchiae to near the end of the body, major spines of chaetiger 5 with a lateral tooth, and no modified posterior notochaetae ( Table 2 ). However, these two species are clearly distinguished by the former having a narrow rounded or weakly incised prostomium instead of a broad bilobed prostomium, no eye spots instead of 4 widely spaced eyespots arranged in a square shape, and major spines of chaetiger 5 with lateral shelf instead of a distinct accessory tooth found in the latter species ( Blake & Maciolek 1987). The bright yellow body color of P. tunicola sp. nov. in life condition ( Fig. 3A–C ) is similar to that of P. aura ( Sato-Okoshi 1998; Sato-Okoshi & Abe 2012), however, these two species are distinguished by the former not having modified posterior notochaetae. The larger anterior spines are the oldest and usually most worn in Polydora species and therefore the lateral tooth is often a worn flange, sheath, or shelf ( Blake 1969; Kudenov 1982; Bertasi 2016). The lateral shelf on the unworn newer posterior spines characterizes the major spines of the new species. Polydora tunicola sp. nov. is unique among the genus Polydora and the family Spionidae because they have tunic-boring symbiotic relationships with ascidians.

Etymology. The specific name tunicola is a combination of two Latin words, namely, tunica and -cola (= dweller), which refers to the peculiar tunic-boring habit of the species. The Japanese name Hoyano-poridora is a combination of the Japanese words hoya (=ascidian), no (case particle), and poridora (= Polydora ), meaning “ Polydora of the ascidian”.

Habitat. Five to 40 m depth of subtidal zone of rocky shores.

Distribution. Currently, it is known from Izu-Oshima Island, Sagami Sea, central Japan and Sakai fishing port, Minabe Town, Wakayama Prefecture, Japan. Additionally, although we have not examined the specimen and the locality is unknown, palps of spionids probably attributable to this species were in a photograph of Polycarpa cryptocarpa kroboja in a Japanese field guidebook ( Nishikawa 2006).

Reproduction and larval morphology. Egg strings and egg capsules were not found. Three-chaetiger larvae were found in the host ascidians during the extraction procedure of adult specimens ( Fig. 3E ). All observed larvae had eye spots and long larval chaetae.

Host association. Currently, Polycarpa cf. cryptocarpa kroboja and Cnemidocarpa sp. ( Ascidiacea: Styelidae ) are known as hosts. All specimens of P. tunicola sp. nov. burrow into the tunica and construct mud tubes on the host surface ( Fig. 4A, B ). The positions of the burrows are variable, but most worms construct burrows with the openings near siphons of host ascidians ( Fig. 4C, D ). One juvenile worm, which did not bore but constructed a tube at the tip of the host siphon, was observed in December 2017 ( Fig. 4E ). The number of worms per host was zero to several tens of individuals. The second author (OH) has been observing marine organisms by more than 500 dives per year for more than 20 years in the sea of Izu-Oshima Island, but worms matching the new species of Polydora were not observed so far in other substrates including other ascidians (e.g., Clavelina cyclus Tokioka & Nishikawa, 1975 , Cnemidocarpa irene ( Hartmeyer, 1906) , Didemnum vexillum Kott, 2002 , Diplosoma mitsukurii Oka, 1892 , Polyandrocarpa misakiensis Watanabe & Tokioka, 1972 , Polycitor proliferus ( Oka, 1933) , Pseudodistoma fragile Tokioka, 1958 , Pyura mirabilis ( Drasche, 1884) , Rhopalaea circula Monniot & Monniot, 2001 , Styela clava Herdman, 1881 , and Botryllinae spp.).

Anatomical ( Fig. 4F ) and micro-CT ( Fig. 5 ) examination of the ascidians showed that P. tunicola sp. nov. bored into the cellulose-rich ascidian tunics and constructed a U-shaped burrow ( Fig. 5A, B ). In the cross-sectional images of the host, many pairs of the boreholes of P. tunicola sp. nov. were observed in the host tunica because the burrows were U-shaped and the two apertures were adjacent to each other ( Fig. 5C–E ). Polydora tunicola sp. nov. constructed the external mud tubes on the host surface as extensions of the two openings of the burrow (i.e., approximately twice as many external mud tubes as burrows) and the burrows were lined with mud and silt.

Molecular phylogeny. The intraspecific variation in gene sequences of five P. tunicola sp. nov. specimens were 0% (0/1749 bp) for 18S, 0%–0.13% (0–1/767 bp) for 28S, and 0%–0.42% (0–2/473 bp) for 16S rRNA. The phylogenic analyses of the concatenated sequences recovered P. tunicola sp. nov. as the sister species to P. aura within a well-supported clade ( Fig. 6 ). Polydora tunicola sp. nov. and P. aura also formed a well-supported clade with P. lingulicola and P. cf. glycymerica .

Cellulase activity. The results of plate assay ( Fig. 7A ) and zymography ( Fig. 7B ) consistently detected the cellulase activities in the individuals of P. tunicola sp. nov. Zymography showed multiple forms of cellulase enzymes with molecular weights near 63 and 50 kDa.

Figures

FIGURE 2. Polydora tunicola sp. nov. Light micrographs showing the morphology of preserved specimens (holotype: NSMT- Pol H-855). A: entire body. B: anterior end, dorsal view. C: anterior end, lateral view. D: posterior end, lateral view. E: posterior end, dorsal view. F: posterior end, ventral view. Scale bars: (A) = 5 mm; (B–C) = 1 mm; (D–F) = 500 μm.

FIGURE 3. Polydora tunicola sp. nov. Light micrographs showing the morphology of living (A–D) and fixed (E–I) specimens. A–D, F–I: paratypes (NSMT-Pol P-856), E: non-type larvae. A: anterior end, dorsal view. B: anterior end, lateral view. C: 2–11 chaetigers, lateral view. D: glandular pouches in the 7th to 14th chaetigers. E: larvae from a maternal tube. F: notopodial falcate spines and companion chaetae of the 5th chaetiger. G: neuropodial hooded hooks of 7th chaetiger. H: hooded hooks from neurochaetae of 20th chaetiger. I: hooded hooks from neurochaetae of posterior (60th) chaetiger. br—branchia; ca—caruncle; cc—companion chaetae; gp—glandular pouch; hh—hooded hook; lt—lateral tooth; ma—median antenna; fs—falcate spine; nec—neuropodial capillaries; nel—neuropodial lamella; noc—notopodial capillaries; nol—notopodial lamella; pa—palp; pr— prostomium; sh—shelf; vc—ventral capillaries. Scale bars: (A–D) = 1 mm; (E) = 300 μm; (F–I) = 50 μm.

FIGURE 4. Polydora tunicola sp. nov. A: palps and mud tubes of Polydora tunicola sp. nov. protruding from their burrows in the tunics of the sessile ascidian Polycarpa cf. cryptocarpa kroboja. B: closeup of palps and mud tubes of Polydora tunicola sp. nov. C, D: tubes of Polydora tunicola sp. nov. concentrated on near siphons of the ascidians. E: juvenile of Polydora tunicola sp. nov. (arrowhead) constructing a mucous tube on the siphons of the ascidians but not yet bore into the tunic. F: cross-section of a host ascidian showing many pairs of boreholes filled with mud tubes (arrowheads) of Polydora tunicola sp. nov. in the host tunic. Photo by O. Hoshino (A–E) and H. Abe (F).

FIGURE 5. Micro-CT images of the host ascidian. A: three-dimensional (3D) reconstructed image of three U-shaped burrows of Polydora tunicola sp. nov. B: two-dimensional reconstructed images of three U-shaped burrows of Polydora tunicola sp. nov. extracted from tomographic data. Arrowheads of figures A and B indicate the apertures of the burrows. C–E: Cross-sectional image of the host. Arrowheads indicate the boreholes of Polydora tunicola sp. nov. in the ascidian tunic. Scale bars = 10 mm.

FIGURE 6. Maximum likelihood tree inferred from concatenated sequences of nuclear 18S and 28S and mitochondrial 16S rRNA gene sequences of Polydora species obtained in this study and from DDBJ/EMBL/GenBank database (Table 1). The gene sequences obtained in this study are highlighted in boldface. SH-aLRT/approximate Bayes support/ultrafast bootstrap support values of ≥80%/≥0.95/≥95%, respectively, are given beside the respective nodes. Nodes with red circles indicate triple high sup- port values of SH-aLRT ≥ 80, approximate Bayes support ≥ 0.95, and ultrafast bootstrap support ≥ 95. The scale bar represents the number of substitutions per site. Sequences of Dipolydora species are used for outgroup rooting. The symbols at the left of the species names indicate the lifestyle based on the available information (Table 1).

FIGURE 7. Results of plate assay (A) and zymographic analysis (B) for cellulase activity of Polydora tunicola sp. nov. Haloes (A: destained areas) and destained bands (B: arrowheads) indicate the cellulose decomposition (exhibition of cellulase activity). PC = positive control (crystalline style of a brackish water clam Corbicula japonica), NC = negative control.

Tables

TABLE 2. Morphological characteristics of known Polydora species which have a single median antenna on the caruncle and chaetiger 5 without dorsal superior capillaries, but

Species	Prostomium	Caruncle	Branchiae	Chaetiger 5		Modified	Pygidium	Pigmentation	Life style	Reference
		(maximal	from	Major spine	Companion	posterior
		length)	chaetiger 7 to		chaetae	notochaetae
Polydora tunicola	Rounded or	End of	Near the end	Falcate with	Present	Absent	Flare-disc with	Yellowish	Tunic-borer	This study
Abe, Hoshino &	weakly incised	chaetiger 3		lateral tooth or			mid-dorsal	pigment
Yamada, sp. nov.				shelf			gap; rimmed	especially
							with black	anterior and
								posterior
								chaetiger
P. aura	Incised	End of	Near the end	Falcate with	Present	Present	Wide flaring	Brown and	Shell-borer	Sato-Okoshi
Sato-Okoshi, 1998		chaetiger 2–3		lateral flange			disc	orange pigment		(1998);
								on body		Sato-Okoshi &
										Abe (2012)
P. cornuta Bosc,	Bifurcated	End of	Near the end	Falcate with	Present	Absent	Flaring cup	Usually black	Tube-	Radashevsky
1802	and flaring	chaetiger 3		lateral tooth			to disc with	spots on	dweller	(2005)
	laterally			and subdistal			middorsal	lateral sides of
				flange or keel			gap to narrow	chaetigers from
							incision	7–10 to 10–19
P. fusca	Incised	End of	About 3/4	Falcate with	Present	Present	Cup-shaped	Scattered black	Tube-	Radashevsky &
Radashevsky &		chaetiger 2	of body	small lateral			with dorsal	pigment on head	dweller	Hsieh (2000)
Hsieh, 2000				tooth			gap	and body
P. glycymerica	Incised	Middle of	Near the end	Falcate with	Present	Absent	Disc-like with	Absent	Shell-borer	Radashevsky
Radashevsky, 1993		chaetiger 2		lateral flange or			wide dorsal			(1993)
				sheath			gap
P. latispinosa	Incised	End of	Continuing	Falcate with	present	Present	Large flaring	Brown pigment	Shell-borer	Blake & Kudenov
Blake & Kudenov,		chaetiger 2	to posterior	lateral sheath or			disc with wide	along edges of		(1978)
1978			end	flange			dorsal gap	caruncle

......continued on the next page