Trilobodrilus windansea

Habitat Intertidal zone of a clean, coarse, well-sorted sandy beach, 0.5 – 1 m below the mid- and high water mark.

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. 1 A). Body length 715 µm (499 – 1040 µm, n = 12), width 86 µm (84 – 189 µm, n = 12), body segments indistinct (Fig. 1 A – C, Tables 1, 7). Prostomial shape square (Fig. 1 A – F, Table 7). Eyes lacking. Middle of mouth located 110 µm (77 – 133 µm, n = 9) posterior of the apical tip (mo, Fig. 1 A, C – D, Table 1). Four compound cilia terminally on prostomium (pcc, Fig. 1 B, 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. 1 A, D, Table 1). Prostomial compound cilia surrounded by five pairs of semicircularly arranged apical ciliary tufts (act, Fig. 1 B, E – F, Tables 1, 7). Two ciliary bands on prostomium and one ciliary band posterior to the nuchal organs (cb 1 – 3, Fig. 1 A – F, Table 7). First ciliary band (12 – 19 µm wide, n = 3) encircles prostomium with 7 – 8 µm wide dorsal gap (cb 1, Fig. 1 A – B, E – F, Table 7). One pair of intermediate ciliary tufts located laterally between first and second ciliary band (ict, Fig. 1 B, 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. 1 B, 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. 1 E – F, Table 7). One lateral pair of nuchal organs located between the second and third ciliary band (no, Fig. 1 B, E – F). Third ciliary band dorsally incomplete with wide gap (cb 3, Fig. 1 B, 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. 1 B, E). The ventral ciliary tract extends from posterior prostomium to posterior pygidium (vct, Fig. 1 B – 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. 1 G). Epidermal inclusions in the epidermis (Fig. 1 H, 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: 18 S rDNA, MG 588089 (1857 nucleotides (nt), Table 4); 28 S rDNA, MG 588091 (1126 nt, Table 5); COI, MG 588093 (644 nt, Table 6); CytB, MG 588095 (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. 18 S rDNA fragment is 99.9 % similar to the 18 S rDNA of T. ellenscrippsae sp. nov., and 99.1 % (T. nipponicus) – 99.5 % (T. axi) similar to the 18 S rDNA fragments of the other sequenced species (Table 4). Its 28 S rDNA fragment is 99.9 % similar to the 28 S rDNA of T. ellenscrippsae sp. nov., and 98.6 % (T. axi) – 99.4 % (T. nipponicus) similar to the 28 S 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. 1, Tables 1, 3 – 7

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 A 8207, SIO-BIC A 8208, NHMD- 210468). Additional specimens mounted on slides were unfortunately lost after conducting the measurements.

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.

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.

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).

Distribution Trilobodrilus windansea sp. nov. is only known from Windansea Beach, La Jolla, San Diego, California.

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 A 8206). 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 A 8207, SIO-BIC A 8208, NHMD- 210468). Additional specimens mounted on slides were unfortunately lost after conducting the measurements.