Two new meiofaunal species of Trilobodrilus (Dinophilidae, Annelida) from California, USA Author Kerbl, Alexandra Author Vereide, Emilie Hernes Author Gonzalez, Brett C. Author Rouse, Greg W. Author Worsaae, Katrine text European Journal of Taxonomy 2018 421 1 18 journal article 30189 10.5852/ejt.2018.421 b5331c96-ecb8-49aa-a7dd-545949989adf 1228304 BC3D3D2B-7EBB-4A5C-8FB8-25D33EA4E524 Trilobodrilus windansea sp. nov. urn:lsid:zoobank.org:act:A9DB66C3-C548-45C1-98F6-6EFBA019CF40 Fig. 1 , Tables 1 , 3–7 Diagnosis Trilobodrilus with two pairs of long prostomial compound cilia (1.5–2 times longer than locomotory cilia), encircled by five pairs of intermediate ciliary tufts anterior to the prostomial ciliary bands. First ciliary band with small dorsal gap. Dorsally incomplete second ciliary band with mid-dorsal ciliary tuft and additional posterior ciliary row. Dorsally incomplete third ciliary band posterior to nuchal organs, elongated epidermal inclusions, no spindle glands. Fig. 1 (next page). Trilobodrilus windansea sp. nov. A, D, G, H. Light micrographs; B, D-F. SEM micrographs of several adult specimens. A . Adult live specimen in dorsoventral view. B . Adult specimen in lateral view. C . Adult specimen in ventral view. D . Detail of the prostomium of a live specimen in dorsoventral view. E . Detail of prostomium and anterior body region in lateral view, with the insert showing the additional ciliary row posterior to the second ciliary band. F . Detail of the prostomium in dorsal view. G . Detail of the posterior body region of an adult live female with two eggs. H . Detail of the dorsal epidermis of an adult live specimen with epidermal inclusions, one of them enlarged in insert. Abbreviations: acf = anterior ciliary field; acr, additional ciliary row; act = apical ciliary tuft; cb1–3 = ciliary bands 1–3; egg = eggs; ei = epidermal inclusion; ict = intermediate ciliary tuft; mdt = middorsal ciliary tuft of the second ciliary band; mo = mouth opening; no = nuchal organ; np = neuropil; pcc = prostomial compound cilia; phb = pharyngeal bulb; pyg = pygidium; sto = stomach; vct = ventral ciliary tract. All images oriented with the anterior tip to the left. All scale bars = 50 µm, if not denoted otherwise. Table 4. Comparison of similarities between 18S gene fragments in species of Trilobodrilus Remane, 1925 . For accession numbers of the used sequences see Table 3.

T. windansea	T. ellenscrippsae	T. itoi	T. nipponicus	T. axi
sp. nov.	sp. nov.
T. windansea sp. nov.
T. ellenscrippsae sp. nov.	0.999
T. itoi	0.991	0.991
T. nipponicus	0.990	0.991	0.993
T. axi	0.952	0.953	0.948	0.950
T. heideri	0.955	0.955	0.951	0.953	0.982

Table 5. Comparison of similarities between 28S gene fragments in species of Trilobodrilus Remane, 1925 . For accession numbers of the used sequences see Table 3.

T. windansea sp. nov.	T. ellenscrippsae sp. nov.	T. itoi	T. nipponicus	T. axi
T. windansea sp. nov.
T. ellenscrippsae sp. nov.	0.996
T. itoi	0.936	0.937
T. nipponicus	0.944	0.943	0.969
T. axi	0.850	0.850	0.855	0.858
T. heideri	0.859	0.860	0.862	0.863	0.901

Table 6. Comparison of similarities between COI gene fragments in species of Trilobodrilus Remane, 1925 . For accession numbers of the used sequences see Table 3.

T. windansea sp. nov.	T. ellenscrippsae	T. itoi
sp. nov.
T. windansea sp. nov.
T. ellenscrippsae sp. nov.	0.849
T. itoi	0.786	0.760
T. nipponicus	0.769	0.760	0.776

Table 7 (next page). Morphological characters and their states in all described species of Trilobodrilus Remane, 1925 . Measurements are given in mm for body length and width, and in µm for the size of epidermal inclusions; the latter are taken from Kajihara et al. (2015) , and from the original sources (Rao 1973; Remane 1925; Riser 1999; Uchida & Okuda 1943; Westheide 1967, 2008). Abbreviations: G = gonochoristic; H = hermaphroditic.

Species	T. windansea	T. ellenscrippsae	T. heideri	T. axi	T. hermaphroditus	T. indicus	T. nipponicus	T. itoi
sp. nov.	sp. nov.	Remane, 1925	Westheide, Riser, 1999 1967		Rao, 1973	Uchida & Okuda, 1943	Kajihara, Ikoma, Yamasaki &
Hiruta, 2015
Body length [mm]	0.5–1.0	0.5–0.7	1.5–1.9	1	1.7	1.0–1.2	0.7–1.4	0.9–1.3
Body width [mm]	0.08–0.2	0.09–0.2	0.1–0.2	0.1	0.22	0.1–0.12	0.09–0.16	0.08–0.12
Prostomial shape	Squared	Conical	Squared	Conical	Conical	Conical	Conical	Squared
Pairs of apical ciliary tufts	5	4	?	?	?	?	2–3	3–4
Mid-dorsal tufts on 2nd ciliary	1	1	0	0	0 (but 2 stereocilia)	0	1	1
band (#)
Interm. ciliary tufts (#)	present (1)	present (>2)	? (absent)	? (present)	?	?	present (1)	present (>2)
Add. ciliary row(s) posterior of 2nd ciliary band	present	absent	present	present	absent	?	absent	absent
3rd ciliary band dorsally	absent	present	absent	absent	present (but not	present	present	absent
continuous	meeting ventral
ciliary tract)
4th ciliary band	absent	present	absent	present	?	?	present	present
Segmentally arranged lateral	absent	present (not as	?	?	present	? (absent)	present	present
ciliary tufts	regular as in other
species)
Epidermal inclusions [µm × µm] 4 × 9		?	?	10 × 15	6 × 9	3 × 12	5.5 × 16	7 × 9
Spherules/ envelope (#)	4–8	?	3–10	5–19	3–7	7–11	9–13	3–22
Length/ width of epidermal inclusions (mean)	2.2	?	1.41	1.81	1.42	2.42	2.91	1.29
Spindle glands	absent	?	present	present	present	present	absent	absent
Sex	G	G	G	G	H	G	G	G
Habitat	intertidal	subtidal	subtidal	intertidal	intertidal/ subtidal	intertidal	intertidal	intertidal
Type locality	Windansea	La Jolla Cove, La	Helgo-land,	Sylt,	Nahant Bay, Palm Beach, Akkeshi Bay,			Ishikari Beach,
Beach, La Jolla, Jolla, California,		Germany	Germany	near middle of	Andhra	Japan	Japan
California, USA USA 9 m depth		shor of Nahant,	Pradesh,
Massachusetts, USA India
6 m depth
Source	This study	This study	Remane (1925)	Westheide	Riser (1999)	Rao (1973) Uchida & Okuda Kajihara et al.
(1967)	(1943)	(2015)

Etymology This species is named after the beach where it was collected, Windansea Beach. Material examined Holotype UNITED STATES OF AMERICA : complete adult, 805 µm long (platinum-palladinium-coated and mounted on stub for SEM), Windansea Beach , La Jolla , San Diego , California , 32°49′46″ N , 117°16′51″ W , coarse sand in holes at 0.5 m and 1 m at the mid ‐ and high ‐water mark, respectively , 14 Apr. 2009 , K Worsaae and G Rouse leg. ( SIO-BIC A8206 ). Paratypes UNITED STATES OF AMERICA : 4 specimens (2 mounted on same stub as holotype for SEM, 2 in 70% EtOH), same locality and sampling site as the holotype , 14 and 17 April 2009 (SIO-BIC A8207, SIO-BIC A8208, NHMD- 210468 ). Additional specimens mounted on slides were unfortunately lost after conducting the measurements. Description Measurements given from holotype , ranges given in parenthesis include paratypes and lost specimens. Transparent body, light brown to dark brown tint (live and fixed specimens) ( Fig. 1A ). Body length 715 µm (499–1040 µm, n = 12), width 86 µm (84–189 µm, n = 12), body segments indistinct ( Fig. 1A– C , Tables 1 , 7 ). Prostomial shape square ( Fig. 1A–F , Table 7 ). Eyes lacking. Middle of mouth located 110 µm (77– 133 µm, n = 9) posterior of the apical tip (mo, Fig. 1A, C–D , Table 1 ). Four compound cilia terminally on prostomium (pcc, Fig. 1B, E–F ), each consisting of approximately 50–60 cilia (n = 3), spaced 32 µm apart (18–32 µm, n = 10). Compound cilia substantially (up to two times) longer than locomotory cilia in ciliary bands (19–43 µm relative to 8–20 µm in ciliary bands, n = 10, Fig. 1A, D , Table 1 ). Prostomial compound cilia surrounded by five pairs of semicircularly arranged apical ciliary tufts (act, Fig. 1B, E–F , Tables 1 , 7 ). Two ciliary bands on prostomium and one ciliary band posterior to the nuchal organs (cb1–3, Fig. 1A–F , Table 7 ). First ciliary band (12–19 µm wide, n = 3) encircles prostomium with 7–8 µm wide dorsal gap (cb1, Fig. 1A–B, E–F , Table 7 ). One pair of intermediate ciliary tufts located laterally between first and second ciliary band (ict, Fig. 1B, E ). Second ciliary band (13–19 µm wide) dorsally incomplete with 43–46 µm wide gap and one mid-dorsal ciliary tuft in center of gap (mdt, Fig. 1B, E–F , Table 7 ; consisting of 50–70 cilia, n = 3). One additional thin row of cilia (5–7 µm wide, n = 2) lines the second ciliary band postero-laterally, not extending dorsally for the entire length of the second ciliary band (acr, Fig. 1E–F , Table 7 ). One lateral pair of nuchal organs located between the second and third ciliary band (no, Fig. 1B, E–F ). Third ciliary band dorsally incomplete with wide gap (cb3, Fig. 1B, E–F , Table 7 ); width of third ciliary band 10–17 µm, width of gap 39–50 µm (n = 3). Individual cilia scattered across the body ( Fig. 1B, E ). The ventral ciliary tract extends from posterior prostomium to posterior pygidium (vct, Fig. 1B–C, E ; width of tract relative to total body width approximately 0.6 (n = 3)). Anus opening dorso-anteriorly on pygidium. Eggs 126–143 µm long, located 534–464 µm posterior to the prostomial tip and 209–194 µm (n = 2) anterior to pygidial tip ( Fig. 1G ). Epidermal inclusions in the epidermis ( Fig. 1H , Table 7 ); average measurement of envelope 4 × 9 µm, 4–8 spherules per envelope (n = 12); no spindle glands. Molecular information The following sequences were determined by standard sequencing from a single, non-type specimen collected on 14 Apr 2009 , for which no morphological voucher remains: 18S rDNA, MG588089 (1857 nucleotides (nt), Table 4 ); 28S rDNA, MG588091 (1126 nt, Table 5 ); COI, MG588093 (644 nt, Table 6 ); CytB, MG588095 (421 nt). In the following, the sequences of T. windansea sp. nov. are first compared to the most similar sequences found in T. ellenscrippsae sp. nov. , and subsequently the range of similarities to the respective species is listed. Trilobodrilus windansea sp. nov. 18S rDNA fragment is 99.9% similar to the 18S rDNA of T. ellenscrippsae sp. nov. , and 99.1% ( T. nipponicus ) – 99.5% ( T. axi ) similar to the 18S rDNA fragments of the other sequenced species ( Table 4 ). Its 28S rDNA fragment is 99.9% similar to the 28S rDNA of T. ellenscrippsae sp. nov. , and 98.6% ( T. axi ) – 99.4% ( T. nipponicus ) similar to the 28S rDNA fragments of the other sequenced species ( Table 5 ). COI is 84.9% similar to the COI of T. ellenscrippsae sp. nov. , and 76.9% ( T. itoi ) – 78.6% ( T. nipponicus ) similar to the sequences of the other species ( Table 6 ). Cytochrome B is 85.6% similar to T. ellenscrippsae sp. nov. In both the maximum-likelihood as well as the Bayesian analyses, T. windansea sp. nov. was found to be the sister species of T. ellenscrippsae sp. nov. with full support, the two of them forming the sister group to the T. itoi – T. nipponicus clade ( Fig. 2 ). These Pacific species were shown to be the sister clade to the group formed by the Atlantic species T. axi and T. heideri ( Fig. 2 ). All Trilobodrilus -species form a sister-clade to Dinophilus sp., a representative of the second dinophilid genus ( Fig. 2 ). Fig. 2. Phylogenetic tree resulting from Bayesian analysis based on combined COI, 18S, and 28S rRNA gene sequences of already known and sequenced species and the newly described species of Trilobodrilus Remane, 1925 . Numbers above nodes indicate maximum-likelihood bootstrap support values in percent (> 60%), and numbers below nodes indicate posterior probabilities from Bayesian inference (> 0.90), respectively. The yellow box labels the two species from the West Coast of the USA, the orange box the species from Japan, which are collectively labelled by the red box as Pacific species. The green box labels species recorded from the Atlantic, and all these species form the clade Trilobodrilus (violet box). Habitat Intertidal zone of a clean, coarse, well-sorted sandy beach, 0.5–1 m below the mid- and high water mark. Distribution Trilobodrilus windansea sp. nov. is only known from Windansea Beach, La Jolla, San Diego, California . Remarks Trilobodrilus windansea sp. nov. most closely resembles T. itoi in morphology (but T. ellenscrippsae sp. nov. in its molecular profile), but still differs by having more apical ciliary tufts, by having an additional row of cilia posterior to the second ciliary band, and by lacking a fourth ciliary band as well as segmentally arranged ciliary tufts along the body ( Fig. 1 , Table 7 ). Furthermore, the epidermal inclusions are more elongated and include fewer spherules than in T. itoi ( Table 7 ). Trilobodrilus windansea sp. nov. resembles the other Californian species, T. ellenscrippsae sp. nov. , in having a dorsally incomplete second ciliary band with mid ‐dorsal tuft, but differs in having longer prostomial compound cilia, one more pair of apical ciliary tufts, a small dorsal gap in the first ciliary band, an additional row of cilia posterior to the second ciliary band, a dorsally incomplete third ciliary band, and by lacking a fourth ciliary band ( Fig. 1 , Table 7 ). Trilobodrilus windansea sp. nov. further differs from T. ellenscrippsae sp. nov. , T. itoi , T. axi and T. heideri in comparison of gene fragments, see Molecular information above or Tables 4–6 .