Two new meiofaunal species of Trilobodrilus (Dinophilidae, Annelida) from California, USA

. We describe two new species of the annelid genus Trilobodrilus Remane, 1925 (Dinophilidae Verill, 1892) from an intertidal and a subtidal location in San Diego, California. These two species show morphological and molecular divergences between each other and the previously described, geographically distant species. Intertidal T. windansea sp. nov. differs from subtidal T. ellenscrippsae sp. nov. most remarkably in the number and pattern of ciliary tufts and bands on the prostomium and along the body length, besides showing ca. 15% difference in gene fragments of COI and CytB. Trilobodrilus windansea sp. nov., though nesting with T. ellenscrippsae sp. nov. in the molecular phylogenetic analyses, morphologically resembles the Japanese T.

The distribution of the six described species of Trilobodrilus shows regional overlap, but with differences in habitat: Trilobodrilus heideri Remane, 1925 is found in the German North Sea (Helgoland, Sylt) and in the Roscoff-area, where it co-occurrs with T. axi Westheide, 1967, though the former is always found subtidally and the latter intertidally (Remane 1925;Westheide 1967Westheide , 2008;;Ax 1968).Trilobodrilus heideri is also reported from the British coast at Anglesey, from Skagerak (Swedish West Coast, Gullmarfjord) and the Mediterranean Sea (Boaden 1963(Boaden , 1966;;Westheide 2008).Trilobodrilus axi is known from the coasts of Arcachon, France, and Massachusetts, USA, where this intertidal species cooccurs with the inter-and subtidal species T. hermaphroditus (Westheide 1967(Westheide , 2008;;Riser 1999).The intertidal species T. itoi Kajihara, Ikoma, Yamasaki &Hiruta, 2015 andT. nipponicus Uchida &Okuda, 1943 are both found in Hokkaido, Japan, but in different bays along the coast (Uchida & Okuda 1943;Kajihara et al. 2015).Trilobodrilus nipponicus was also possibly found in Seattle, USA, representing the single report of the genus from the west coast of the USA (Wieser 1957).Trilobodrilus indicus Rao, 1973 was described from the intertidal zone in the Bay of Bengal, India (Rao 1973).
Due to the limited number of obvious external characters, detailed morphological examinations of Trilobodrilus are necessary for species identification.Given that all Dinophilidae have six inconspicuous body segments and that all species of Trilobodrilus are of comparable size and shape, the number and continuity of ciliary bands, number and arrangement of ciliary tufts, and the shape and size of epidermal inclusions have been shown to be the most taxonomically informative morphological characters (Westheide 1967(Westheide , 2008;;Kajihara et al. 2015).
The here presented study describes two species of Trilobodrilus from coastal southern California, based on light and electron microscopic examinations as well as on molecular sequencing and a phylogenetic analysis of the genus.

Sampling
Specimens were extracted from sediment samples collected at two different locations in San Diego County, California, USA, during April 2009; intertidally at Windansea Beach, La Jolla from holes dug 0.5 m and 1 m deep at the mid-and high-water marks in coarse sand, and subtidally using scuba diving, sampling the surface of silty shell gravel at 9 m depth off La Jolla Cove, La Jolla.All specimens were extracted by MgCl 2 anaesthetization with decantation and sorted using dissecting microscopes.Prior to fixation, all specimens were re-anaesthetized in a 1:1 solution of seawater and isotonic MgCl 2 and fixed either in paraformaldehyde (PFA, 3.7% in phosphate buffered saline (PBS)) or trialdehyde (3.7% paraformaldehyde/ 4% glutaraldehyde in 0.1 M sodium cacodylate buffer) overnight at 4°C.Subsequently, specimens fixed in paraformaldehyde were rinsed several times in PBS and stored with 0.05% NaN 3 , while samples fixed in trialdehyde were transferred to 0.1 M sodium cacodylate buffer.Specimens for molecular analyses were stored directly in 99% ethanol.

Light microscopy (LM)
Live animals were observed, photographed, and video recorded using a UI-1540 camera mounted on a Leica DMR compound microscope (with DIC).Eleven fixed specimens of T. windansea sp.nov.and four fixed specimens of T. ellenscrippsae sp.nov.were mounted individually in dorso-ventral orientation (whenever possible), and investigated using an Olympus IX 70 inverted compound microscope.Images were obtained using a mounted Olympus DP73 camera in combination with the CellSens Entry software package v. 1.6.Morphological measurements (Table 1) were conducted using either the previously mentioned software package or by using ImageJ v. 1.47 (Schneider et al. 2012).Two specimens were afterwards transferred to EtOH for preservation (T.windansea sp.nov.paratypes: SIO-BIC A8208, NHMD-210468), but the remaining specimens were unfortunately lost in the process and are not preserved.

Scanning electron microscopy (SEM)
Three specimens of T. windansea sp.nov.and one specimen of T. ellenscrippsae sp.nov., which were fixed in trialdehyde and stored in 0.1 M sodium cacodylate buffer, were postfixed in an aqueous 2% OsO 4 solution for 1 h at room temperature (RT) and subsequently rinsed in demineralized water.Following dehydration via an ascending ethanol series and transferring to acetone, samples were critical-point dried (using an Autosamdri 815-machine at the Natural History Museum of Denmark, University of Copenhagen) and mounted on aluminum stubs.A high-resolution sputter coater (JFC-2300HR) applied an approximately 80-90 nm layer of platinum/palladium-mixture onto the samples prior to examination using a JEOL JSM-6335F field emission scanning electron microscope at the Natural History Museum of Denmark, University of Copenhagen. 1) from SEM were scaled and measured using ImageJ v. 1.47 (Schneider et al. 2012).

Morphological measurements (Table
All holotypes and several paratype specimens are deposited at the Scripps Institution of Oceanography, San Diego, California, USA (SIO-BIC A8206-SIO-BIC A8209).One paratype of T. windansea sp.nov.(NHMD-210468) is deposited at the Natural History Museum in Copenhagen, Denmark.

DNA extraction, amplification, and sequencing
DNA extractions were performed using a Qiagen DNeasy Tissue and Blood kit (Qiagen, Düsseldorf, Germany) following the manufacturer's protocol.DNA elution was in 80-90 μL of buffer, and was repeated with the original buffer volume to maximize DNA yield.Genes used to reconstruct the phylogeny included 18S rRNA, 28S rRNA, cytochrome c oxidase subunit I (COI), and cytochrome B (CytB).Specific primer details, including references, are listed in Table 2.All PCR specifications, including visualization, purification, and sequencing follow that of Martínez et al. (2015) and Gonzalez et al. (2017).
Chromatogram reading and contig assembly were performed in Sequencher 4.10.1 (GeneCodes Corporation, Ann Arbor, MI, USA).All chromatograms were blasted to check for contamination using  (Hall 1999).All newly generated sequences were deposited in GenBank ® with the accession numbers MG588089-MG588096 (Table 3).
Sequences of 18S rRNA, 28S rRNA, and COI were aligned using the MAFFT online platform (Katoh & Standley 2013), with sequences of 18S rRNA and 28S rRNA being aligned using the interactive refinement algorithm L-insi with the parameter 'nwildcard' selected (Katoh et al. 2005), while COI was aligned using the 'Auto' strategy.All genes were concatenated using Sequence Matrix (Vaidya

Phylogenetic analyses
Molecular datasets were analysed with maximum likelihood (ML) and Bayesian methods (BA).Phylogenetic analyses were run on the CIPRES Science Gateway (Miller et al. 2010), and specific phylogenetic parameter settings for ML and BA followed that of Gonzalez et al. (2017).jModelTest (Posada 2008) was used to infer optimal evolutionary models on alignments of individual genes estimated by the corrected Akaike information criterion (AICc) prior to BA.Both 18S rRNA and COI were analysed under a GTR model with gamma distribution and a proportion of invariable sites (GTR + I + Γ), while 28S rRNA was analysed under a GTR model with gamma distribution (GTR + Γ).
Pairwise similarities of individual genes were obtained using MEGA-7 (Kumar et al. 2016) and are listed in 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.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 1967Westheide , 2008)).Abbreviations: G = gonochoristic; H = hermaphroditic.

Etymology
This species is named after the beach where it was collected, Windansea Beach.

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.

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.
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).

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).
Additional specimens mounted on slides were unfortunately lost after conducting the measurements.

Description
Measurements given first in the text from holotype, ranges in in parenthesis include measurements taken from the lost specimens.
No eggs present in the investigated specimens.
Epidermal inclusions and spindle glands in the epidermis could not be described and measured due to insufficient preservation in the investigated specimens.

Molecular information
The following sequences were determined by Sanger sequencing from a single, non-type specimen collected on 19 April 2009, for which no morphological voucher remains: 18S rDNA, MG588090 (1857 nucleotides (nt), Table 4); 28S rDNA, MG588092 (1126 nt, Table 5); COI, MG588094 (644 nt, Table 6); CytB, MG588096 (426 nt).In the following, the sequences of T. ellenscrippsae sp.nov.are first compared to the most similar sequences found in T. windansea sp.nov., and the range of similarities with the addition of the respective species are subsequently listed.

Fig. 1 (
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.

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).

Fig. 3 (
Fig. 3 (next page).Trilobodrilus ellenscrippsae sp.nov.A, F. Light micrographs; B-E.SEM micrographs of several adult specimens.A. Adult fixed specimen in dorsoventral view, pygidium missing.B. Adult specimen in lateral view.C. Adult specimen in ventral view.D. Apical view of the prostomium of an adult specimen.E. Detail of the prostomium and anterior body region of and adult specimen in lateral view.F. Detail of the prostomium of a fixed adult specimen in dorsoventral view.Abbreviations: acf = anterior ciliary field; act = apical ciliary tuft; cb1-4 = ciliary bands 1-4; ict = intermediate ciliary tuft; lct = lateral ciliary tuft; mdt = mid-dorsal 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 1 .
Measurements and morphological characters of holotype, as well as minimum, maximum and average of all measured T. windansea sp.nov.and T. ellenscrippsae sp.nov.The number of measured specimens is added to the table, all measurements are either given as µm (length or width), as distances (measured in µm from the anterior tip), or as relative measurements; "n/a" is used for measurements which are not applicable, since epidermal inclusions could not be detected in fixed material of T. ellenscrippsae sp.nov.
NCBI BLAST.Sequences were visualized and trimmed with BioEdit

Table 2 .
List of primers used for amplification and sequencing, with original references.

Table 3 .
Listet al. 2011).Sequences of CytB (only available from the two here described Trilobodrilus species) were aligned with Clustal W in BioEdit.

Table 4 .
Remane, 1925.similarities between 18S gene fragments in species of TrilobodrilusRemane, 1925.For accession numbers of the used sequences see Table3.

Table 5 .
Remane, 1925.similarities between 28S gene fragments in species of TrilobodrilusRemane, 1925.For accession numbers of the used sequences see Table3.

Table 6 .
Remane, 1925.similarities between COI gene fragments in species of TrilobodrilusRemane, 1925.For accession numbers of the used sequences see Table3.

Table 7 (
next page).Morphological characters and their states in all described species of Trilobodrilus DiagnosisShort Trilobodrilus with two pairs of prostomial compound cilia encircled by four pairs of intermediate ciliary tufts anterior to first ciliary band.Additional ciliary tufts between first and second ciliary band.Dorsally continuous third ciliary band.Incomplete fourth ciliary band, posterior of third ciliary band with broad dorsal gap. -7