﻿Ribbon worms (phylum Nemertea) from Bodega Bay, California: A largely undescribed diversity

﻿Abstract The diversity of nemerteans along the Pacific coast of the United States is regarded as well characterized, but there remain many cryptic, undescribed, and “orphan” species (those known only in their larval form). Recent sampling of nemerteans in Oregon and Washington has begun to fill in these taxonomic gaps, but nemertean diversity in California has received relatively little attention over the past 60 years. During the summers of 2019 and 2020, nemertean specimens were collected from 20 locations in the Bodega Bay region of northern California, USA, including rocky intertidal shores, sandy beaches, mudflats, and other habitats. Based on morphological assessment and DNA sequence analysis (partial Cytochrome Oxidase I and 16S rRNA genes), our surveys identified 34 nemertean species. Only 13 of these (38%) can be confidently assigned to described species. Another 11 represent species that are new to science, including members of the genera Riserius, Nipponnemertes, Poseidonemertes, Zygonemertes, Nemertellina, Oerstedia, and three species of uncertain affiliation. The remaining ten species include undescribed or cryptic species of uncertain status that have been found previously along the Pacific Coast of North America. Our surveys also document extensions of known geographic ranges for multiple species, including the first records in California of Antarctonemertesphyllospadicola, Cephalothrixhermaphroditica, and Maculauraoregonensis. This is the first report of the genus Nemertellina in the northeast Pacific and Riserius in California. Overall, our findings highlight how much remains to be learned about the diversity and distribution of nemerteans in the northeast Pacific.

Nemerteans are challenging to identify given their relatively small number of external features, many of which do not fall into discrete character states (e.g., color and shape of body), and the fact that many species have been described and are known only from histological study of formalin-preserved material.The phylum is known to possess large numbers of cryptic species, that is, morphologically indistinguishable, but nevertheless distinct, species (e.g., Chen et al. 2010;Leasi and Norenburg 2014;Hao et al. 2015;Hiebert and Maslakova 2015a).Given that morphological approaches often fail to distinguish between look-alikes, DNA-based approaches are increasingly used to identify and delimit species, resolve phylogenies, and infer biogeographical patterns (e.g., Chernyshev et al. 2021a;Mendes et al. 2021;Kajihara et al. 2022aKajihara et al. , 2022b)).
The nemertean fauna of the Cold Temperate Northern Pacific province (as per Spalding et al. 2007) is among the best-characterized in the world, as it has been studied by several nemertinologists during the course of the past century and a half (e.g., Stimpson 1857;Griffin 1898;Coe 1901Coe , 1904Coe , 1905Coe , 1940;;Corrêa 1964).However, new nemertean species are routinely discovered by researchers working in this region as molecular techniques are applied, and as new locations, life stages, habitats and depths are sampled and living specimens studied (e.g., Maslakova and von Dassow 2012;Hiebert et al. 2013;Hao et al. 2015;Hiebert andMaslakova 2015a, 2015b;Hunt and Maslakova 2017;Chernyshev et al. 2021b).Roe et al. (2007) report 65 intertidal nemertean species for the Oregonian Biogeographic Province (Oregon to Central California).However, recent sampling and DNA-barcoding of adults and planktonic larvae increased the known number of nemertean species in Oregon alone to more than a hundred (Hiebert 2016;Maslakova et al. 2022).In contrast, the diversity of nemerteans in California has received relatively little attention during the past 60 years, and molecular methods have not been applied broadly to the nemertean fauna of California.
The Bodega Bay region in northern California supports biodiverse ecosystems including rocky shores, kelp forests, sandy beaches, seagrass beds, and mudflats.The diversity of nemerteans in the Bodega Bay region has received some attention historically.A student report by Tamura (1957) identified 12 nemertean species in this region.Corrêa (1964) surveyed the southern end of Bodega Bay, including Dillon Beach, Tomales Point, and Tomales Bay, and identified 18 nemertean species.A pair of surveys conducted of Bodega Harbor (Standing et al. 1975) and the open coast of Bodega Marine Reserve (Ristau et al. 1978) identified seven nemertean species.Collectively, these previous studies identified a total of 21 different species of nemerteans in this region.The goal of this study was to extend the geographic scope of recent investigations of nemertean diversity in the northeast Pacific (Hiebert 2016;Maslakova et al. 2022) to include northern California.In particular, we collected nemerteans from a variety of habitats in the Bodega Bay region and used DNA barcoding to examine this fauna for the first time.

Specimen collection and preservation
During the summers of 2019 and 2020, we collected samples from 20 locations around Bodega Bay, California (Fig. 1, Tables 1, 2).
We selected the locations to encompass a variety of habitat types, including rocky substrate and soft sediments in both wave-exposed/outer coast and wave-protected, estuarine/harbor environments.Most of our collections were from the intertidal zone, but some subtidal collections were made as well.In the field, we haphazardly searched for visible nemerteans and additionally collected complex habitat material to extract small worms in the laboratory.Complex material (e.g., sedentary/colonial animals, algal holdfasts, surfgrass roots, sand, mud, etc.) was collected in the field and placed into sealed plastic bags.In the laboratory, we transferred these materials into clear aquaria, and covered the material with seawater.These samples were left for several days and checked regularly to remove any nemerteans observed crawling on the walls or water's surface.After several days, we broke apart and sorted through complex habitat material to remove hidden worms.Colonial ascidians and other invertebrates from samples of benthic communities tended to deteriorate rapidly in the laboratory and caused any nemertean specimens to become unusable, so these materials were sorted soon after collection.Photographs and videos of worms were taken using a Leica MC170HD digital camera mounted to a Leica M125 dissecting microscope or a Leica DM1000 compound microscope, with accompanying software (Leica Application Suite v. 4.4).For hoplonemerteans, the stylet and basis of living specimens were photographed, when possible, using a compound microscope.We made initial species identifications using available morphological keys and geographically relevant inventories of Nemertea (Coe 1905;Roe et al. 2007;Hiebert 2016).Tissue samples were then preserved in 95% ethanol for DNA extraction, and some specimens were preserved in 4% formalin as morphological vouchers.Specimens collected in 2019 were identified with numbers preceded by the letter "B" and those collected in 2020 were given identifiers beginning with the label "BON."Morphological and tissue vouchers have been deposited in the National Museum of Natural History, Smithsonian Institution, Washington, D.C. (Table 2, see also BOLD dataset https://dx.doi.org/10.5883/DS-NEMBBCA).

DNA extraction, PCR, sequence analysis, and species delimitation
We extracted DNA from 76 individuals using DNEasy Blood and Tissue Kit (Qiagen) following the manufacturer's protocol.We attempted to PCR-amplify portions of two mitochondrial genes, cytochrome c oxidase subunit I (COI) and 16S rRNA, from each individual, using universal and nemertean-specific primers (Table 3).
Each PCR was performed in a 20 μl volume, with 1 unit per reaction of Go Taq Polymerase (Promega) with supplied buffer, 200 μM dNTPs, and 500 nM of each primer.We used the following thermocycle profile: 95 °C 2 min; 34 cycles of: 95 °C 40 s, 45 °C (COI) 48-50 °C (16S) 40 s, and 72 °C 1 min; followed by final extension for 2 min (72 °C).Some DNA extracts required dilution (~ 1:20) for PCR success.PCR products were assessed with gel electrophoresis, purified with Wizard SV Gel and PCR Clean Up Kit (Promega), and sequenced at Sequetech (Mountain View, CA) in both directions using PCR primers.We used Geneious Prime for sequence analysis.Sequences with initial HQ < 50% were discarded.Each sequence was manually trimmed to eliminate primers and low-quality end regions.Forward and reverse strands were aligned, proofread against each other using quality PHRED scores and chromatograms, and contigs used to generate consensus sequences.Nucleotides with combined PHRED score of less than 20 in consensus sequences were trimmed off or converted to "N".We translated each COI nucleotide sequence into amino acids using the Invertebrate Mitochondrial translation table and checked for the presence of stop codons.
Consensus sequences were checked against the NCBI database (GenBank) using nucleotide BLAST to screen for contamination and to aid with specimen identification.A 4% p-distance divergence was previously identified as appropriate for species delineation in large scale COI-barcoding studies of nemerteans (e.g., Kvist et al. 2014;Maslakova et al. 2022), and thus was used here as a criterion for selection of conspecific reference sequences.When available, conspecific sequences from GenBank were added to the alignment.Reference sequences from closely related species were added where no conspecific reference sequences were available (Suppl.material 1).All newly generated sequences have been deposited in BOLD and GenBank (Table 2, see also BOLD dataset https://dx.doi.org/10.5883/DS-NEMBBCA).We aligned sequences using the MAFFT plug-in within Geneious, using default parameters, visually inspected alignments for gaps and irregularities, then used them to construct unrooted neighbor-joining trees (Tamura Nei substitution model).Final COI alignment

Results
Of the 76 specimens from which DNA was extracted, we were able to successfully obtain sequences from 68; a total of 64 COI sequences and 57 16S sequences (see Table 2, BOLD dataset https://dx.doi.org/10.5883/DS-NEMBB-CA).Results of the ASAP analysis of the COI data suggested the presence of a barcoding gap (i.e., the separation between the maximum intraspecific and the minimum interspecific variation) between 3-5%, similar to previous large-scale DNA-barcoding studies of nemerteans (e.g., Kvist et al. 2014;Sundberg et al. 2016;Maslakova et al. 2022).Using 0.038 simple p-distance as the threshold, ASAP partitioned the dataset into 52 OTUs, 33 of which contained all our samples (Table 4).BOLD species delineation algorithm partitioned our sequences into 34 barcode index numbers (BINs, Table 4), splitting Zygonemertes sp.BOBA015 into two separate BINs.Of the 34 BINs, ten are unique, meaning they lacked representation in BOLD (likely not previously sequenced).We were not able to obtain a COI sequence from specimen BON41, but its identity was determined morphologically as Micrura wilsoni (Coe, 1904) and was confirmed by the 16S rRNA sequence data.There is no evidence of cryptic species of M. wilsoni in the region thus far.Hence, we report a total of 34 putative species of nemerteans from Bodega Bay, CA (Table 4).COI sequences were obtained for 33/34 species, and 16S sequences for 28/34 species.Neighbor-Joining trees of the COI and 16S rRNA sequence alignments are shown on Figs 2, 3, respectively.Because of the ubiquitous presence of cryptic species among nemerteans, species distributions listed below refer to reports verified by DNA sequence data, unless otherwise noted.Undescribed species, as well as species of uncertain status, were assigned temporary alphanumeric OTU codes (e.g., BOBA0XX) for tracking purposes, until their taxonomy is resolved.

Class PALAEONEMERTEA Order Archinemertea Iwata, 1960 Family Cephalotrichidae McIntosh, 1873 Genus Cephalothrix Örsted, 1843
A species-rich genus of mostly white, thread-like worms, which have a long pre-oral region and lack ocelli as adults.Given their general lack of distinguishing features, the species therein are difficult to differentiate morphologically    (e.g., Chen et al. 2010;Leasi and Norenburg 2014;Kajihara 2019;Sagorny et al. 2019).COI sequences are available for at least ten species from Oregon and California, most of which have not been described and some are only known in their larval form (Hiebert 2016;Maslakova et al. 2022).
Identification.Our specimens share high sequence similarity (99-100% COI) with specimens reported as C. hermaphroditicus from Chile (KU840171), France (MH681952), Spain (KM230034), and Argentina (KM230037).The type locality of the species is Cocholgue, Chile, but given the comparatively low haplotype diversity in Chile, and high haplotype diversity in France, Sagorny et al. (2019) suggested the species may have been introduced to Chile from Europe.COI barcodes for the Bodega Bay specimens are identical to the Chilean ones, consistent with the idea of a single introduction to the Pacific from the Atlantic.
Habitat.Collected from wave-exposed, rocky intertidal habitats, among colonial ascidians.Stations 10, 15, 18 (Fig. 1, Table 1).Notes.The only available sequences of Cephalothrix hermaphroditica from Chile were collected ~ 1,000 km north of its type locality.Given that biogeographical patterns among nemertean species are variable, we tentatively include the species as synonymous with the species we encountered in our surveys.This is the first report of the species in the Cold Temperate Northern Pacific, and first 16S barcode for the species.Cephalothrix hermaphroditica provides the only known example of hermaphrodism among the Palaeonemertea.Reproductive features were not observed by us.

A B D C
Morphology.Filiform body, ~ 50 mm long, pale yellowish to orange, color brighter in the head and foregut regions, paler and somewhat translucent posteriorly (Fig. 4B).
Identification.Our specimens share high (99.6%COI) sequence similarity to the topogenetype of C. simula (GU726622), and other C. simula sequences, as defined by Kajihara et al. (2013).The type locality of C. simula is Japan, but it has since been reported from the Pacific coast of the U.S., the Atlantic, and the Mediterranean.Remarkably, and in contrast to many other reported cases of such widespread distribution among nemertean species, these reports refer to the same species, as assessed by genetic data (Chen et al. 2010;Kajihara et al. 2013;Sagorny et al. 2019).This suggests that C. simula has been broadly introduced to many geographic regions.
Habitat.Notes.Previously reported from southern California (Chen et al. 2010), this is the first report of the species from northern California.Following the detection of C. simula in the Mediterranean, the species was identified as non-native (Fernández-Álvarez and Machordom 2013), likely introduced to locations outside of East Asia in the 21 st century via transport of larvae and juveniles in the ballast water of ships (Chernyshev 2014) or with oyster aquaculture (e.g., Ruesink et al. 2005).Apart from being one of the few known cases of species introductions amongst the nemerteans (but see Moore et al. 2001), C. simula has attracted interest for the high levels of neurotoxin tetrodotoxin (TTX) found in its tissues, sparking concern about potential contamination of shellfish (e.g., Kajihara et al. 2013;Turner et al. 2018).
Morphology.Eyeless white worm 65-87 mm long (Fig. 4C); spatulate head demarcated from the body by width and an indistinct transverse furrow; the mouth a small slit just posterior to the furrow on the ventral side.With large, oblong, conspicuous lateral sense organs located posterior to the tubulanid ring.
Identification.Specimens at hand most resemble Carinomella lactea described by Coe (1905) from San Pedro, California and, to a lesser extent, Tubulanus pellucidus (Coe, 1895).The latter was described from the coast of New England, but later also reported from Monterey Bay, San Pedro, and San Diego, California by Coe (1905).In his description of the species, Coe (1905) notes that T. pellucidus often co-occurs with C. lactea in southern California, but the former is smaller in size (10-25 mm long).The species encountered here did not locate any close sequence matches within GenBank, and there are no previously published sequences of C. lactea.A COI sequence (OK081469) derived from a small unpigmented tubulanid reported from Oregon by Maslakova et al. (2022: OTU 89), is distinct from those of Bodega Bay specimens, and so is the only available COI sequence (HQ848625) purported to belong to an Atlantic T. pellucidus, from North Carolina (Andrade et al. 2012).The 16S sequences place Bodega Bay specimens within a clade of other unpigmented tubulanids including species of Tubulanus, Carinella, and Parahubrechtia (Fig. 3).
Habitat.Mudflats.Collected among polychaete tubes on intertidal mudflats, just below the surface of the sediments (station 8).
Distribution.Bodega Bay, CA, USA (this study).The species has been reported from the Atlantic (Corrêa 1961), but it is unlikely this report refers to the same species.
Notes.Species not previously sequenced.
Identification.Morphology agrees with Tubulanus sexlineatus, a species described from Puget Sound, Washington and Alaska, and DNA sequences show high percent similarity to previously published sequences of T. sexlineatus.Also resembles T. punctatus (Takakura, 1898) from Japan, and T. superbus (Kölliker, 1845) from the Mediterranean.Two described Tubulanus species from the northeast Pacific with similar coloration have no published sequences but can be differentiated from T. sexlineatus by pattern: T. cingulatus (Coe, 1904) is deep brown, with four dorsal longitudinal white lines, and T. capistratus (Coe, 1901) is brown with many narrow white rings, and only three white longitudinal lines (Coe 1901;Corrêa 1964).
Distribution.Discovery Island, BC, Canada (QHAK2597-22, QHAK2649-22 in BOLD); Puget Sound, WA, USA (Thollesson and Norenburg 2003 Cerebratulus is one of three non-monophyletic mega-genera in the Class Pilidiophora, with Lineus and Micrura (e.g., see Kajihara et al. 2022b).The morphological diagnosis of the genus is based on combinations of non-unique characters (Schwartz 2009).However, since Cerebratulus marginatus is the type species of the genus, and the species below is closely related (Verdes et al. 2021), it will likely retain the generic affiliation.

Cerebratulus sp. BOBA008
Cerebratulus marginatus: Andrade et al. 2012: 6;Thollesson and Norenburg 2003: 408;Roe et al. 2007: 224, pl. 88 Morphology.Large (25 cm long), dorsoventrally flattened dull reddish brown worm with lateral margins distinct both in color (pale) and shape (flattened).Spade-shaped head bordered by deep cephalic slits, with a long slit-like mouth located posterior to their endings on the ventral side (Fig. 5C).Capable of swimming.Young individuals have a pair of inconspicuous ocelli near the anterior tip of the head.
Identification.A common intertidal mudflat species, previously reported from Washington and Oregon as Cerebratulus marginatus or Cerebratulus cf.marginatus (Thollesson and Norenburg 2003;Roe et al. 2007;Andrade et al. 2012;Hiebert 2016;Maslakova et al. 2022).The type locality of C. marginatus is Naples, Italy, but the closest available sequenced specimens are from Spain.Although the nominal species C. marginatus is reported from the eastern and  Habitat.Intertidal mudflats in areas with sandy sediments; often > 30 cm below the surface (station 5).
Identification.Resembles Kulikovia spp., and several other species in the Siphonenteron clade (see Lineidae incertae sedis below).COI sequences show that it is conspecific (99-100% similarity) with one of two sister lineages previously reported from southern Oregon in their larval form as Lineus sp. 2 by Hiebert (2016) and as Lineus sp. by Maslakova et al. (2022: OTU 49) in both larval and adult form.Phylogenetic analysis based on five genetic markers places this species within the Siphonenteron clade, more specifically within the genus Kulikovia (Chernyshev et al. 2018;Kajihara et al. 2022b).Two other members of the genus are reported from the northeast Pacific: K. montgomeryi from Kachemak, Alaska and San Juan Island, Washington (as Cerebratulus montgomeryi in Schwartz 2009; Hiebert 2016), and an undescribed species reported from southern Oregon as Lineus sp.'red' by Hiebert and Maslakova (2015b) and as Kulikovia sp. by Maslakova et al. (2022: OTU 59).
Notes.This is the first record of the species in California.

Genus Maculaura Hiebert & Maslakova, 2015a
The genus Maculaura was erected for five cryptic species occurring in NE Pacific that were previously recognized under the name Micrura alaskensis Coe, 1901.A phylogenetic analysis by Kajihara et al. (2022b) shows Maculaura as a well-supported lineid clade.
Morphology.Body 15-18 mm long.Anterior tip white, with lateral cephalic slits, no ocelli, and pink cerebral ganglia visible through the body wall (Fig. 5D).One individual quite pink, with color beginning near the brain, another white throughout the foregut region, with brown coloration beginning in the intestinal area.
Collected from the open coast among the roots of surfgrass (Phyllospadix sp., station 12) and in mid-intertidal mussel beds (station 16).In southern Oregon, this species has also been found within estuaries, especially under rocks at the edges of mudflats (Hiebert and Maslakova 2015a).

BIN. BOLD:ADM2641.
Material examined.B1.Morphology.Body ~ 18 mm long; anterior tip white, eyeless, with lateral cephalic slits, the rest of the body pink.Anterior tip rounded in extension, head not demarcated from the body.Cerebral ganglia rosy and visible through the body wall (Fig. 5E).Body somewhat transparent.Posterior end with translucent, somewhat thickened caudal cirrus.
Habitat.Collected from Bodega Harbor within the holdfast of subtidal Giant Kelp (Macrocystis pyrifera) at a depth of 3-4 m (station 6).In Oregon, individuals have also been found intertidally in sand and mud (Hiebert and Maslakova 2015a).
Notes.This is the first record of the species in California.

Genus Riserius Norenburg, 1993
Riserius is the only known genus of Pilidiophora to exhibit a mesopsammic lifestyle, i.e., living interstitially among sand grains.Its members display a curious suite of features: lack of cutis, cerebral organs opening via lateral pits rather than longitudinal slits, and a transverse cephalic furrow that encircles the body in front of the mouth.Possesses a unique sock-like pilidium recurvatum larva (Hiebert et al. 2013).A single species, Riserius pugetensis Norenburg, 1993, is described from coarse marine sediments of Puget Sound, Washington.Two additional, undescribed species, R. sp."eyes" and R. sp."no eyes," are known from southern Oregon, and two others, R. sp. 3 and R. sp. 4, from the Sea of Japan (Vostok Bay, Russia), all collected exclusively in larval form, but some raised in the laboratory to sexual maturity (Hiebert et al. 2013;Hiebert 2016).Similar larval forms are reported from other parts of the world, but sequence data are lacking (reviewed in Hiebert et al. 2013).Phylogenetic analysis by Kajihara et al. (2022b) suggests Riserius is a derived lineid that lost the lateral cephalic slits.
Morphology.Thread-like cream-colored worm, 50-100 mm long (Fig. 5A), with a long and narrow pointed head not well demarcated from the body, except by the cerebral organ pits.With four ocelli in a small cluster on each side of the head just in front of the pink tinged cerebral ganglia (Fig. 5B).Cerebral organs just posterior to the brain, opening via conspicuous lateral pits.With a V-shaped transverse furrow located posteriorly ~ 3/4 of the length from the tip of the head to the cerebral organs.
Identification.Morphology of our specimens agrees with that of Riserius spp.(Norenburg 1993;Hiebert et al. 2013), though specimens observed here are much longer than R. pugetensis, which is ~ 15 mm.Sequences from Bodega Bay specimens are distinct from those of the previously reported species, and 16S rRNA tree places these individuals within a monophyletic Riserius clade (Fig. 3).Specimens from Bodega Bay represent a new species of Riserius.
Habitat.Collected on wave-exposed, sandy beaches from among very coarse sand in the low intertidal zone at ~ -0.15 m to +0.30 m above Mean Lower Low Water (MLLW, station 13).Small numbers of individuals were also observed on other local beaches among very coarse sand (station 20).
Notes.Species new to science.First record of the genus in California.

LINEIDAE incertae sedis
Lineus and Micrura are non-monophyletic mega-genera within the family Lineidae (e.g., see Kajihara et al. 2022b).The morphological diagnoses of these genera are based on combinations of non-unique characters (Schwartz 2009).
Here we use names Lineus and Micrura as taxonomic artifacts, and not to imply shared common ancestry.
BIN. BOLD:ADS0049.Material examined.BON6, BON11, BON40, BON50, BON60, BON86.Morphology.Body 8-52 mm long, with significant variation in color: pale (semi-transparent), tan, reddish brown, and rosy-orange varieties observed (Fig. 5K), with lighter coloration ventrally, and smaller individuals appearing paler in color.Head not demarcated from the body, with pale margins, and 3-7 red, purple, or black ocelli arranged closely in a single row on each side of the anterior tip along the head margin (Fig. 5L, M).In some individuals, there is an irregular transverse band of white pigment granules just posterior to and between the two rows of ocelli (Fig. 5M).Cerebral ganglia are rosy, but this is faint in some individuals.Mouth just posterior to the end of the lateral cephalic slits on the ventral side.Body tapers posteriorly.
Identification.The six individuals included here match closely (99-100% similarity, COI) to Lineus flavescens reported from southern Oregon (Hiebert 2016;Maslakova et al. 2022: OTU 45) and agree with Coe's description of the species.A closely related species (5-6% divergence, COI) is reported from Dutch Harbor, Alaska (OR590584), Puget Sound, Washington (BBPS027-19 in BOLD) and Charleston, Oregon (Fig. 2; Maslakova et al. 2022: OTU 46).The two species closely resemble each other, but one is known to occur from Alaska to Oregon, while the other from Puget Sound, Washington to Long Beach, California.Because the type locality of L. flavescens Coe, 1904 is San Pedro, California, we assume that we encountered the "real" L. flavescens, and not its more northerly look-alike.

BIN. BOLD:ADW4746.
Material examined.BON2.Morphology.Body ~ 12 cm long, margins and ventral surface white, dorsally with an orange patch at the anterior tip, bordered posteriorly by white, and followed by a broad purple stripe, which is interrupted at intervals by thin, transverse white lines (Fig. 5H).With lateral cephalic slits and a slender caudal cirrus.
Identification.Morphologically, specimens from Bodega Bay resemble Lineus striatus briefly described by Griffin (1898) from Puget Sound, Washington and Micrura verrilli described by Coe (1901) from Prince William Sound, Alaska.In addition, three similar species have been reported from the western Pacific: Micrura bella (Stimpson, 1857), Micrura impressa (Stimpson, 1857), and Micrura festiva Takakura, 1898.Coe synonymized Griffin's taxon with his own (despite Griffin's having priority), and M. impressa and M. festiva and have been treated as synonyms of M. bella (Crandall and Norenburg 2001), although M. impressa is still listed as an accepted species in WoRMS (WoRMS Editorial Board 2023).ASAP analysis of the COI data suggests that all available M. verrilli sequences from the west coast of USA and Canada comprise a single OTU (Fig. 2).A closely related species is reported by Chernyshev and Polyakova (2022) from the Bering Sea as Evelineus sp., but there is no mention of its appearance, and it has only been encountered at depths 350 m and below.COI sequences suggest that Bodega Bay specimens are conspecific (97-99% similarity COI) with M. verrilli reported from Bamfield, BC, Canada (EF125007), Puget Sound, Washington (KF935508), and Charleston, Oregon (Maslakova et al. 2022: OTU 65).One additional available COI sequence (EF125001) of M. verrilli from Puget Sound, Washington contains many ambiguities, which causes some algorithms (e.g., Geneious Prime distance calculations) to interpret it as substantially different from the others, however ASAP analysis places it within the same OTU.A COI sequence of M. bella reported from the Sea of Japan (NC_027727) is ~ 10% different from that of M. verrilli, and thus belongs to a separate species.
Crandall and Norenburg (2001) suggest the extent of the anterior orange patch may help differentiate the eastern (exclusively dorsal) and western (extends onto ventral side) Pacific forms, however one of the M. verrilli specimens found in Washington had pigmentation on both sides (Schwartz 2009).
Habitat.Collected from the low intertidal zone among colonial ascidians (station 18) and observed among kelp holdfasts washed ashore in the Bodega Bay region.
Notes.The species reported here belongs to a clade of lineids with orange or magenta red anterior tip that may be synonymous with Evelineus (Schwartz 2009), also referred to as "lineid lineage A" (Kajihara et al. 2022b) and is not closely related to the type species of the genus, Micrura fasciolata Ehrenberg, 1828 (Chernyshev et al. 2018;Chernyshev and Polyakova 2019).

BIN. BOLD:ADW9830.
Material examined.BON41.Morphology.Dark brown worm, ~ 45 mm long, slightly paler ventrally, cephalic lobe bordered by white at the anterior tip and along the lateral cephalic slits; thin white transverse bands at irregular intervals along most of the body length (Fig. 5G).No ocelli; rosy cerebral ganglia visible through the body wall.With a small white caudal cirrus.
Identification.Conforms to the description of Micrura wilsoni (Coe, 1904), described from Monterey and San Pedro, California.No look-alikes are currently known in the northeast Pacific.Although we were not able to obtain a high-quality COI sequence, 16S rRNA sequence from the Bodega Bay individual is 99-100% identical to those of M. wilsoni reported by Hiebert (2016) from southern Oregon.These individuals correspond to the COI-delimited M. wilsoni of Maslakova et al. (2022: OTU 90).
Habitat.Collected from kelp holdfasts (Macrocystis pyrifera) in the subtidal (station 6) and very low intertidal zones (station 7), also among holdfasts of subtidal bull kelp (Nereocystis luetkeana) washed ashore.In southern Oregon found on the exposed rocky shore under boulders and in rock crevices of the low intertidal zone.
Notes.According to a recent phylogenetic analysis of the family Lineidae this species is not closely related to the type species of the genus, Micrura fasciolata, but is a member of a clade called "lineid lineage G" by Kajihara et al. (2022b).
Material examined.BON59.Morphology.Body 97 mm long, uniformly orange (Fig. 5I).Head the same width as the body, with pale margins, lateral cephalic slits.Ocelli arranged in two rows, one along each anterolateral margin (Fig. 5J), ~ 7 ocelli each, but it is difficult to know the true number as the pigment granules appear broken up and irregular.
Habitat.Collected from a wave-exposed, rocky intertidal habitat among colonial ascidians and coralline algae (station 15).
Notes.Species new to science.

Class HOPLONEMERTEA Hubrecht, 1879
Order Monostilifera Brinkmann, 1917 Suborder Cratenemertea Familial classification suspended as per Kajihara, 2021 Genus Nipponnemertes Friedrich, 1968 Of the seven Nipponnemertes species described from the northeast Pacific, N. bimaculata (Coe, 1901), N. drepanophoroides (Griffin, 1898), N. fernaldi Iwata, 2001, N. occidentalis (Coe, 1905), N. pacifica (Coe, 1905), N. punctatula (Coe, 1905), and N. rubella (Coe, 1905), only two have been reported and/or barcoded in recent years, N. bimaculata and N. punctatula.The type locality of the latter species is southern California, but the only available barcodes are from Japan (Thollesson and Norenburg 2003;Hookabe et al. 2022), so the two may represent distinct species.An unknown species was reported from southern California (Andrade et al. 2012), from 360-390 m, but its identity and relationship to the above species is not yet known.Clearly, there is a diversity of Nipponnemertes species along the Pacific coast of North America, including several cryptic species.Hookabe et al. (2022), in their revision of the genus that includes descriptions of ten new species, reported three clades of Nipponnemertes that are supported by molecular data as well as morphology: degree of head demarcation and presence/absence of a cephalic patch.The species we encountered at Bodega Bay most closely resembles N. bimaculata (Coe, 1901), which belongs to Clade B of Hookabe et al. (2022), with the northwest Pacific species N. jambio Hookabe et al., 2022, N. neonilae Hookabe et al., 2022, N. ojimaorum Hookabe et al., 2022, N. crypta Hookabe et al., 2022, and a potential trans-Pacific complex of species currently known as N. punctatula (Coe, 1905).Species in this complex vary in the degree of development of dorsal and cephalic pigmentation, but most have more or less distinct cephalic pigment patch(es).
Morphology.Body 55-67 mm long, broad, reddish brown dorsally, much paler (almost white) ventrally.Head white, pointed, narrower than the body, with two maroon, triangular pigment patches placed symmetrically on either side of a mid-dorsal ridge (Fig. 6A).Numerous large ocelli lie in the space between the anterolateral margins and the pigment patches, ~ 20 per side.With two pairs of cephalic furrows.The anterior, cerebral organ furrows are equipped with numerous secondary furrows, as is characteristic of other members of the genus (Fig. 6A, inset).The posterior neck furrow forms a posteriorly directed V on the dorsal surface.Pink cerebral ganglia are discernible from the ventral side, just posterior to the cerebral organ furrows (Fig. 6A, inset).Proboscis and stylet apparatus not observed.
Identification.The specimens from Bodega Bay conform to the description of Nipponnemertes bimaculata (Coe, 1901) except for the shape of the cephalic patches, which are triangular in our specimens as opposed to oval in the original description.However, Coe (1905) later reported triangular patches in other specimens.COI sequences from Bodega Bay individuals form a separate OTU from those of the species previously reported as N. bimaculata from Washington and Oregon (Thollesson and Norenburg 2003;Maslakova et al. 2022: OTU 28), as well as an additional species collected in Puget Sound, Washington by CIE (BHFL_5974, Fig. 2), which can be distinguished from other northeast Pacific look-alikes by mottling on the dorsum.The N. bimaculata reported from Washington and Oregon seems to have narrower cephalic patches than the species from Bodega Bay.We have not observed any individuals with oval cephalic patches.The type locality of N. bimaculata is Alaska and Puget Sound, Washington (Coe 1901), although the species was later reported south to Ensenada, Mexico (Coe 1940).We suggest to reserve the name N. bimaculata for the northern form with solid dorsal pigmentation (Maslakova et al. 2022: OTU 28), and to treat Bodega Bay specimens as a new species.
Habitat.Collected from the holdfasts of kelp (Macrocystis pyrifera) in the very low intertidal zone (station 7).
Notes.Species new to science.
Identification.Specimens from Bodega Bay conform to the description of Amphiporus imparispinosus Griffin, 1898 from Port Townsend, Washington and Sitka, Alaska.Two similar species have been described from the northeast Pacific: Amphiporus leuciodus Coe, 1901, from Victoria, BC, Canada and New Metlakatla and Glacier Bay, Alaska, and Amphiporus similis Coe, 1905, from Monterey, California, though Coe later treated the former as a synonym (1905), and the latter as a variety (1940) of A. imparispinosus.Subsequent authors retained all three as valid species (Gibson and Crandall 1989;Crandall and Norenburg 2001; WoRMS; but see Roe et al. 2007).Coe (1905) notes that A. imparispinosus has a pink brain, small cerebral sense organs, three accessory stylet pouches, and is longer (to 75 mm) than A. similis, which is 10-15 mm, with a clear brain, large cerebral sense organs, fewer ocelli and two accessory stylet pouches.Griffin (1898) does not mention color of the cerebral ganglia in A. imparispinosus.Maslakova et al. (2022) report, based on COI sequence data, three distinct A. imparispinosus-like species (OTUs 4-6) from the northeast Pacific.One of those (OTU 4) is distributed from Dutch Harbor, AK to Charleston, OR, and overlaps the original range of A. imparispinosus (including samples from Puget Sound, WA), so may represent the true A. imparispinosus.The other two OTUs have not been reported north of Oregon (Maslakova et al. 2022: OTUs 5 and 6).Another look-alike is reported from the Sea of Japan (Chernyshev and Polyakova 2019: MN211508).The pinkish color of cerebral ganglia and the three accessory stylet pouches in our specimens suggests that it is not A. similis, but an undescribed cryptic species.
Morphology.Body 38-70 mm long, white.Head rounded and wider than body (Fig. 6C).Four clusters of ocelli; two rows following the anterolateral margins, and two more located posteriorly and medially, above the colorless cerebral ganglia (Fig. 6D).The posterior clusters of ocelli appear reddish, while the anterior rows appear brown in reflected light.The neck furrow is obvious and forms a dorsal V-shape posterior to the cerebral ganglia.Basis with rounded posterior margin and slight medial constriction.Central stylet equal in length to the basis (Fig. 7B).Proboscis with three accessory stylet pouches.
Identification.See Amphiporus sp.BOBA018 above.The presence of three pouches of accessory stylets and the length of the worms suggest that this is not A. similis, but an undescribed cryptic species.
Habitat.Collected from wave-exposed, rocky intertidal habitats (stations 18, 19), including on holdfasts of the kelp Egregia menziesii and crawling across other low intertidal surfaces.
Notes.This is the first record of the species in California.Morphology.Body long and slender, green dorsally, cream-colored ventrally.Head not especially demarcated from the body.With numerous ocelli distributed along the colorless anterolateral margins of the head.Pink cerebral ganglia.Basis of central stylet much longer than the slightly curved central stylet.

Genus
Identification.Specimens from Bodega Bay conform to the description of Emplectonema viride Stimpson, 1857, as redescribed by Mendes et al. (2021).There are no known look-alikes in the northeast Pacific.For years, the species has been reported as a synonym of its Atlantic look-alike Emplectonema gracile (Johnston, 1837) (e.g., Roe et al. 2007 and references therein).The two species were recently shown to be distinct, the name E. viride resurrected, and the Pacific species redescribed (Mendes et al. 2021).
Habitat.Collected from a mid-intertidal mussel bed (station 1), and commonly observed in many intertidal habitats throughout the Bodega Bay region.Typically associated with acorn barnacles, which it preys upon.
Notes.For photographs of this species, see Mendes et al. (2021: fig. 3)

Genus Ototyphlonemertes Diesing, 1863
Species of Ototyphlonemertes are exclusively mesopsammic, living in the interstices of well-sorted, coarse marine sediments.They are easily distinguished from other small, slender, white eumonostiliferans by the presence of a pair of statocysts in the cerebral ganglia, and the lack of ocelli in adults, but most of the described species appear to represent cryptic species complexes (e.g., Leasi and Norenburg 2014;Leasi et al. 2016).Two species of Ototyphlonemertes with spirally sculpted stylets are described from the Pacific coast of the U.S.: O. americana Gerner, 1969 andO. spiralis Coe, 1940, and a third species, with a smooth stylet, was reported near San Francisco (Roe et al. 2007).However, the Ototyphlonemertes spp. of the Pacific coast are not well sampled, and "forms that key out to either of the known species have a reasonable probability of not being those species" (Roe et al. 2007).
Morphology.Body 3.5 mm long and less than 1 mm wide, white; foregut region transparent, intestinal region cream-colored.Head slightly demarcated from the body by a transverse cephalic groove.With a pair of statocysts, one in each of the two ventral cerebral ganglia.Statocysts of the polygranular type.Basis slender, irregularly cylindrical, and longer than the spirally sculpted central stylet (Fig. 7D).Proboscis diaphragm not especially long, middle chamber bulbous.
Identification.Specimens from Bodega Bay conform to the description of O. americana Gerner, 1969 from Puget Sound, Washington, but DNA sequence data are not available from Puget Sound region to confirm identification.COI sequences from Bodega Bay specimens in this study match closely (> 99% similarity, COI) with a species previously reported from San Diego, California as Ototyphlonemetes lactea by Leasi et al. (2016: KU230123).O. lactea was described from Brazil (Corrêa 1954), but the name was later used to refer to a group of species with similar morphologies and presumed shared ancestry, called phylomorphs (Envall and Norenburg 2001).O. americana belongs to the lactea type, which was later synonymized with the macintoshi type (Kajihara et al. 2018), a decision supported by a multigene phylogenetic analysis (Leasi et al. 2016).Relative to macintoshi type worms, O. americana and O. lactea have a shorter proboscis diaphragm and a bulbous middle chamber, as opposed to a long, tubular one.Another OTU of a lactea type reported from Half Moon Bay, California by Leasi et al. (2016: KU230128) is likely to be confused with the Bodega Bay species.Sampling of Ototyphylonemertes in Puget Sound is needed to resolve the specific identity of these lineages.
Habitat.Collected from low intertidal, coarse marine sediments on a wave-exposed sandy beach (station 13).
Morphology.Body ~ 5 cm long, orangish purple dorsally, paler ventrally.Head slightly demarcated from the body, with red cerebral ganglia visible through the body wall.Stylet apparatus not observed.
Identification.Paranemertes peregrina is a cryptic species complex composed of at least seven distinct lineages (Hao et al. 2015).A single widely distributed lineage likely corresponding to the P. peregrina Coe, 1901 originally described from Alaska has been identified on the basis of proximity to the type locality and habitat, and is confirmed by DNA sequence data to occur from British Columbia, Canada to southern Oregon, USA on the northeast Pacific coast, and from Kuril Islands, Russia to Shandong, China on the northwest Pacific coast (Hao et al. 2015).The remaining six species, including the two previously reported from Bodega Bay, remain undescribed.
Habitat.Collected from kelp holdfasts in shallow subtidal zone within Bodega Harbor (station 6).
Notes.Hao et al. (2015) report that the two Paranemertes cf.peregrina OTUs from Bodega Bay are well-separated by habitat, with one occurring in mudflats and the other in rocky intertidal contexts, under stones, among algae, etc.Interestingly, the species encountered in this survey came from among kelp holdfasts but matches the one previously found in mudflats by Hao et al. (2015).This might not be as surprising as it appears at first because the kelp holdfasts collected by us (station 6) were from a small kelp bed within Bodega Harbor immediately adjacent to extensive mudflats.For photographs of this species, see Hao et al. (2015: fig. 1J).
Habitat.Collected from an intertidal mudflat, just below the surface of the sediment, among polychaete tubes (station 8).
Notes.Species new to science.

BIN. BOLD:ADX0572.
Material examined.BON1.Morphology.Body 27 mm long, brown dorsally, pale ventrally.Head rounded with colorless margins and a broad brown patch, differentiated only slightly from the body by the colorless transverse band (Fig. 6I).With four eyes; the anterior pair halfway between the anterior tip and the cerebral organ furrows, the posterior pair just below the posterior furrow.With bright red blood vessels.Cylindrical basis, slightly longer than the central stylet (Fig. 7F).With two accessory stylet pouches.
Identification.Fits the description of Tetrastemma nigrifrons Coe, 1904, described from Monterey Bay, California.A look-alike, Tetrastemma stimpsoni Chernyshev, 1992 occurs in the northwest Pacific and the Sea of Japan (Chernyshev et al. 2021a).A third, closely related species has been documented from Dutch Harbor, Alaska (6-7% divergence, BIN: BOLD:AEC4254).No pictures are available, but the description ("brown dorsally, white band separates head, 2 prs eye spots") matches that of this species.
Notes.This species has variable coloration, both in terms of pattern and the amount of pigmentation (Roe et al. 2007;Maslakova, unpublished).Some specimens are almost completely dark brown dorsally with a colorless transverse bar separating the cephalic patch from the dorsum, while others have much less dorsal pigment (e.g., it may be separated into two more or less continuous stripes by a pigment-less mid-dorsal region).Some specimens may be almost entirely devoid of pigment, except for the cephalic patch (which may be broken into two by a mid-dorsal gap).With four ocelli.Blood vessels are red, and clearly show through the body wall.
Morphology.Small and slender, transparent, fast-moving worm; internal structures appear yellowish through the body wall.Anterior tip with a small white patch.With four ocelli (Fig. 6J).Stylet apparatus not observed.
Identification.Anterior white patch and otherwise featureless body distinguishes this from other species of Tetrastemma reported from northeast Pacific (Roe et al. 2007).DNA sequences from the Bodega Bay specimen match those of Tetrastemma sp. 1 first reported from southern Oregon by Hiebert (2016) and Maslakova et al. (2022: OTU 19 as Tetrastemma sp.).Two other overall similar species lacking the anterior white patch occur in southern Oregon (Maslakova et al. 2022: OTUs 15 and 20).
Notes.First record of the species in California.This species is very common in southern Oregon.Reproductive individuals were found in July in southern Oregon, and deposited egg masses in laboratory dishes upon collection, with crawl-away juveniles hatching a week or two later.
Material examined.BON75.Morphology.Body 1.7 mm long, transparent, with an orange gut.With four eyes and two pairs of cephalic furrows: cerebral organ furrows at the level of the posterior pair of eyes, and a V-shaped neck furrow posteriorly, overlying the anterior portion of the cerebral ganglia.Conical basis, significantly longer than the central stylet (Fig. 7O).
Identification.Resembles other featureless species of Tetrastemma.COI sequences show it to be distinct from any previously sequenced species.
Habitat.Collected from low intertidal, coarse marine sediments on a wave-exposed sandy beach (station 13).
Notes.Species new to science.

Genus Zygonemertes Montgomery, 1897
Members of the genus Zygonemertes are distinct from other eumonostiliferans in having a single row of post cerebral ocelli on each side, along the lateral nerve cords, in addition to the more typical ocelli found in rows or groups on the head.In addition, all species we have had the opportunity to examine possess sickle-shaped microscopic inclusions in the epidermis, and most have a characteristically truncated basis of the central stylet.
Three species of Zygonemertes are reported from the northeast Pacific coast: Z. albida Coe, 1901, Z. thalassina Coe, 1901, and Z. virescens (Verrill, 1879).The first two were described by Coe from British Columbia and Alaska, respectively.Zygonemertes thallasina has never been reported outside its type locality, Z. albida was subsequently reported by Coe to occur as far south as Ensenada, Mexico (Coe 1944), and Z. virescens is reported to have a very wide geographic distribution including Pacific (British Columbia to Mexico), Atlantic (Maine to Florida) and Gulf coasts of North America, as well as Curaçao (Coe 1940;Gibson 1995;Roe et al. 2007), but clearly represents a large cryptic species complex (Maslakova, unpublished).The type locality of Z. virescens is New England.Presently we are aware of several genetically distinct Atlantic lookalikes (e.g., from Florida, Colombia, and Caribbean Panama); these are also distinct from several Pacific Z. virescens-like forms.Based on this, it seems most reasonable to exclude Z. virescens from the list of Pacific fauna, and to describe the Pacific forms as new species.
Zygonemertes thalassina was regarded as being extremely similar to Z. virescens, except often longer (to 60 mm), darker in color (olive green), with a smaller S/B ratio, a shorter, stubbier central stylet, and with five stylets per accessory pouch, rather than two or three (Coe 1901(Coe , 1905)).Zygonemertes albida was distinguished on the basis of its small size, lack of color, longer proboscis, and differences in the stylet apparatus, appearing similar to juveniles of the other two species.Among individuals of Z. virescens, Coe noted variation in color, number and arrangement of ocelli, and relative proportions of central stylet and basis.While some of these features may be variable (e.g., with age or environment), we consider it likely that he encountered more than one species, as there are at least four Z. virescens-like species in southern Oregon alone (Maslakova et al. 2022: OTUs 23-26), and we identified an additional two species in this study.The increasing number of Zygonemertes species uncovered with genetic data, and the lack of barcodes from type localities make it difficult to assign existing names to these species.For now, we refer to them as Zygonemertes spp.until formal descriptions are made.We consider reports of Z. albida from the Atlantic coast dubious (Zattara et al. 2019).
Morphology.Body somewhat transparent, greenish yellow with clear margins; 15-25 ocelli arranged in two irregular rows on each side of the head, with a single row of post cerebral ocelli on each side along the lateral nerve cords.Cerebral ganglia pink, visible through the body wall (Fig. 6K).With two pairs of cephalic furrows; cerebral organ furrows are simple ventrolateral arches located 1/2 to 2/3 of the way between the anterior tip and the neck furrow, which overlays the anterior portion of the cerebral ganglia.Specimens B5 and B9 had long slender basis with a slightly concave to flat posterior margin, B6 had a flared stylet basis similar to the illustration for Z. virescens in Roe et al. (2007).Central stylet shorter than basis, S/B ~ 0.5 (Fig. 7G); two accessory stylet pouches, with two stylets each.
Habitat.Collected from Bodega Harbor within the holdfasts of subtidal Giant Kelp (Macrocystis pyrifera) at a depth of 3-4 m (station 6).
Notes.Species not previously reported from California.In the first round of PCR with universal primers, we apparently amplified the gut contents of these worms, the barnacle Balanus glandula.Like another barnacle-eating nemertean, Emplectonema viride, some specimens of this species have a long, slender basis of central stylet.The basis in this species appears slightly narrower than in other species of Zygonemertes.BIN.BOLD:ADW7912.Material examined.BON27, BON76, BON80, BON87.
Morphology.Body 4-15 mm long; color ranging from white with a tinge of yellow to orange, sometimes with dark pigment spots along the sides of the body or at the posterior (Fig. 6M).25-50+ ocelli arranged in four irregular rows on the head, a single row of ~ 10 post-cerebral ocelli along the lateral nerve cords (Fig. 6N).With two pairs of cephalic furrows: cerebral organ furrows are simple ventrolateral arches located 2/3 of the distance from the tip of the head to the V-shaped neck furrow, which overlies the cerebral ganglia.Basis slender, much longer than the central stylet, S/B ~ 0.5-0.65,sometimes with slight medial constriction posteriorly, and with flat or slightly concave posterior margin (Fig. 7I).Two accessory stylet pouches with two or three stylets each.
Identification.See above on species of Zygonemertes.COI sequences from Bodega Bay specimens match a subset of those reported as Zygonemertes sp. 1 (Hiebert 2016) and Zygonemertes sp.(Maslakova et al. 2022: OTU 26) from southern Oregon, and two specimens from Dutch Harbor, Alaska.This species may correspond to Z. albida or a cryptic undescribed species.
Notes.First record of the species in California.Reproductive individuals encountered in August in Bodega Bay.
Material examined.B7, B8, BON88, BON91.Morphology.Body up to 31 mm long, greenish yellow, digestive tract appearing bright reddish orange ventrally.With ~ 15 ocelli on each side of the head, arranged in four irregular rows, and a single row of post-cerebral ocelli (~ 8) along each lateral nerve cord (Fig. 6L).Cerebral organ furrows 2/3 the distance between the anterior tip and the posterior V-shaped neck furrow.Cylindrical basis, longer than the central stylet, with truncated posterior margin (Fig. 7H).S/B ~ 0.5-0.65.Two accessory stylet pouches, with two or three stylets each.
Identification.See above on species of Zygonemertes.Habitat.Collected from Bodega Harbor within the holdfasts of subtidal Giant Kelp (Macrocystis pyrifera) in the shallow subtidal zone (< 5 m depth, station 6).
Morphology.Body 4-15 mm long, brownish, with numerous ocelli arranged in four irregular rows on the head, ~ 25+ on each side (Fig. 6O).Basis quite massive compared to central stylet, in terms of length and width (Fig. 7J), though this is not as obvious in smaller specimens (BON63).S/B 0.4-0.75.One individual (BON81) had an unusual triangle-shaped basis, widening significantly posteriorly, with a flat posterior margin.Two accessory stylet pouches with two or three stylets each.
to posterior end of the body.Basis oval, widening a bit posteriorly, with a slender central stylet, S > B (Fig. 7K).With two accessory stylet pouches, with one or two stylets each.
Identification.Specimens from Bodega Bay conform to the description of Antarctonemertes phyllospadicola (Stricker, 1985) described from San Juan Island, Washington, and the COI sequences match those of A. phyllospadicola from San Juan Island, Washington (Thollesson and Norenburg 2003) and southern Oregon (Maslakova, unpublished).
Habitat.Collected with intertidal samples of surfgrass, Phyllospadix scouleri (station 14).In the San Juan Islands, WA this species is found on blades and inside female inflorescences of P. scouleri in the low intertidal zone.
Notes.This is the first record of this genus and species for California.

Genus Nemertellina Friedrich, 1935
The genus Nemertellina has never been reported from the northeast Pacific and currently contains five valid species, three occurring in Kiel Bay, Germany, and one each in Madagascar and Japan.Members of this genus have four eyes, with the anterior and posterior pairs widely separated; small and simple cerebral organs located far anterior to the brain and opening ventrally near the tip of the head; short rhynchocoel; conical or pear-shaped basis, with 2-4 accessory stylet pouches.Nemertellina canea Friedrich, 1935b, N. minuta Friedrich, 1935a, N. oculata Friedrich, 1935band N. tropica Kirsteuer, 1965  Morphology.Body small and slender, ~ 15 mm long, cylindrical in cross-section (Fig. 6Q).Head the same width as adjacent body.Four eyes occupy the corners of a rectangle, the distance between the anterior and posterior pairs of eyes is considerably larger than the distance between the two eyes of each pair.With two pairs of cephalic furrows: cerebral organ furrows are just posterior to the anterior pair of eyes, and the posterior neck furrow overlies the anterior margin of the cerebral ganglia, which are translucent.Stylet basis cylindrical, rounded posteriorly, S/B ~ 1 (Fig. 7L); two accessory stylet pouches.The rhynchocoel extends 3/4 of the body length.
Identification.The species encountered here is most similar to Nemertellina yamaokai in possessing two sets of cephalic furrows.The Bodega Bay specimens are ~ 10% divergent (COI) from N. yamaokai, suggesting the presence of a sixth Nemertellina species, and the first reported from the northeast Pacific.
Habitat.Collected subtidally in Bodega Harbor from kelp holdfasts (station 6) and among fouling organisms on boat marina docks (station 9).Collected intertidally from rocky shores on the open coast among surfgrass roots (station 14).
Habitat.Collected from a wave-exposed mussel bed (station 1) and found in similar habitat in southern Oregon.
Notes.Coe's (1904) original record of the species from San Diego, California is not verified by DNA sequence data, but given the distinctiveness of this species, and the absence of known look-alikes on this coast, the reported distribution seems likely.According to a recent molecular phylogeny of Tetrastemma and its allies (Chernyshev et al. 2021a) this species does not belong to Tetrastemma sensu stricto or the infraorder Amphiporina, but instead is a member of Oerstediina.Its generic placement remains uncertain.

Discussion
The geographic distributions and abundances of coastal species are changing in response to a variety of human impacts (O'Hara et al. 2021), including warming oceans (Sagarin et al. 1999;Sorte et al. 2010;Lonhart et al. 2019;Sanford et al. 2019).Evaluation of these ecological changes is hindered by our incomplete knowledge of the fauna and flora of coastal ecosystems, pointing to a critical need for assessment of biodiversity (Gray 1997).Currently, up to 90% of marine eukaryotic species are estimated to remain undescribed (Mora et al. 2011;Appeltans et al. 2012).Lesser-studied groups, such as nemerteans, have an especially high fraction of undescribed and undetected species (e.g., Hiebert 2016;Maslakova et al. 2022).
Our study extends the geographic focus of recent taxonomic work on nemerteans to include northern California where relatively little work has been done on nemertean diversity during the past 60 years.Notably, only 13 of the 34 species (38%) we collected and barcoded can be unambiguously assigned to described species.This highlights that nemertean diversity remains poorly known in the northeast Pacific despite more than a century of study.That the majority of the observed diversity cannot be assigned to described species renders the few existing geographically relevant identification guides (e.g., Coe 1940;Corrêa 1964;Kozloff 1996;Roe et al. 2007) inadequate.
Eleven species (32%) reported here are new to science, and ten (29%) comprise previously reported undescribed species, or cryptic species whose taxonomic status cannot be resolved with data at hand (Table 4).Some of the newly discovered species were likely overlooked or set aside because of their relatively small size and evident cryptic morphology (e.g., Tetrastemma sp.BOBA020, Nemertellina sp.BOBA011).Others represent cryptic lineages of previously described species (e.g., Amphiporus cruentatus, Amphiporus imparispinosus, Lineus flavescens, Nipponnemertes bimaculata, Oerstedia dorsalis, Poseidonemertes collaris, Zygonemertes virescens).Yet others may have escaped notice due to their rarity or cryptic habits.
Twenty two of the 34 species have not been previously confirmed by DNA barcodes to occur in northern California.This includes two species that appear to have been introduced from other parts of the world (Cephalothrix simula from the northwest Pacific, and Cephalothrix hermaphroditica from European waters, possibly via Chile or another point of entry along the Pacific Coast of the Americas).
In fact, our study is the first to report C. hermaphroditica from the northeast Pacific.Introduction of C. simula may be of concern to aquaculture due to its association with oysters, and high levels of tetrodotoxin in its tissues (Kajihara et al. 2013;Turner et al. 2018).These are the first reports in California of two previously described species, Antarctonemertes phyllospadicola and Maculaura oregonensis, and six previously reported but undescribed species (Kulikovia sp.BOBA003, Amphiporus sp.BOBA018, Tetrastemma sp.BOBA029, Zygonemertes sp.BOBA012, 13, Eumonostilifera sp.BOBA016).This is also the first record of the genus Riserius in California and Nemertellina in the northeast Pacific.
Historical surveys of nemertean diversity in the Bodega Bay region (Tamura 1957;Corrêa 1964;Standing et al. 1975;Ristau et al. 1978) identified 21 different species.The majority of these species were also found in our surveys, although in some cases, taxonomic uncertainty makes a direct comparison difficult.
Several species recorded in historical surveys of the Bodega Bay region were absent from our surveys.For example, Corrêa (1964) reported a single specimen of Lineus pictifrons, a species we did not locate.Corrêa (1964) also reported several species (e.g., Tubulanus cingulatus, Cerebratulus longiceps) that were dredged with soft sediments at 6 m depth in Tomales Bay, a subtidal habitat that we did not survey.Perhaps the most conspicuous absence in our survey was the lack of Tubulanus ruber (Griffin, 1898), which was reported under the name Tubulanus polymorphus in three of the historical studies cited above and was described as "rather common" in the Bodega Bay region by Corrêa (1964).DNA barcodes suggest that T. ruber, originally described from Alaska, is distinct from Tubulanus polymorphus Renier, 1804, an Atlantic species with which it was previously synonymized by Coe (1940) (Hiebert 2016).The geographic range of T. ruber (as T. polymorphus) has been reported as San Luis Obispo, CA to Alaska (Morris et al. 1980), and from Monterey, CA to the Aleutian Islands (Roe et al. 2007).iNaturalist documents only two clear records of Tubulanus "polymorphus" from central California (San Mateo County, iNaturalist 19743045;and San Luis Obispo County, iNaturalist 84371695), and many records from northern California (Mendocino County) to Alaska.While seasonality could potentially explain the absence of some previously documented species in our surveys, one of us (ES) has conducted intertidal fieldwork in this region throughout the year for the past 20 years and has never observed T. ruber in Sonoma County.The scarcity of recent observations of this large and conspicuous red/orange ribbon worm from Bodega Bay and the southern portion of its geographic range is consistent with a geographic range contraction.

Conclusions
Our findings demonstrate how much there is to learn about the diversity and distribution of nemerteans of the northeast Pacific, particularly among southern regions that have received the least amount of attention.Lack of baseline occurrence data (supported by DNA barcodes) hinders our ability to detect shifts in the distribution and abundance of these species.Further sampling and DNA barcoding along the west coast of North America is needed to obtain a more accurate picture of the diversity in this region.Sampling type localities of previously described species will help resolve some of the taxonomic ambiguities associated with species already encountered.
8 5 7 5 C e re b ra tu lu s m a rg in a tu s W A , U S A B 1 M a c u la u ra o re g o n e n s is M T 7 5 0 2 5 5 M a c u la u ra o re g o n e n s is O R , U S A B 1 9 M a c u la u ra c e re b ro s a M T 7 5 0 2 3 5 M a c u la u r a c e r e b r o s a O R , U S A B O N 8 5 M a c u la u r a c e r e b r o s a B O N 8 3 M a c u la u r a c e r e b r o ip h o n e n te ro n g e n .s p .B O B A 0 0 4 B O N 0 6 L in e u s fl a v e s c e n s B O N 8 6 L in e u s fl a v e s c e n s B O N 1 1 L in e u s fl a v e s c e n s B O N 40 Li ne us fla ve sc en s KU 19 77 63 Li ne us fla ve sc en s O R , U SA BO N 60 Lin eu s fla ve sc en s BO N 50 Lin eus flav esc ens KU1 9778 5 Line us flave scen s OR, USA BON 02 Micrura verrilli OK081405 Micrura verrilli OR, USA EF125 007 Micrur a verrilli Bamfie ld, Canad a EF1 250 01 Mic rura verr illi WA , USA AJ 43 69 49 Tu bu lan us se xlin ea tus WA , US A B1 0 Tu bu la nu s se xli ne at us K U 19 77 08 Tu bu la nu s sp . 2 o s e id o n e m e rt e s c o lla ri s C A , U S A B 1 4 P o se id o n e m e rt e s sp .B O B A 0 1 0 B O N 35 P os ei do ne m er te s sp .B O B A 03 3 B2 Pa ra ne m er te s sp .BO BA 00 9 KP 20 00 55 Pa ran em ert es sp .BD G-M1 CA , US A KP 200 047 Par ane me rtes sp.BD G-A 1 CA ,US A KP20 0131 Paran emer tes pereg rina Tofin o, Cana da B5 Zygonemertes sp.BOBA012 OK081456 Zygonemertes sp. 1 OR, USA B6 Zygo neme rtes sp.BOBA 012 BO N 27 Zyg one me rtes sp.BO BA 013 OK 08 17 02 Zy go ne me rte s sp . 1 OR , US A BO N 76 Zy go ne m er te s sp .BO BA 01 3 B O N 80 Z yg on em er te s sp .B O B A 01 3 B O N 8 7 Z yg o n e m e rt e s sp .B O B A 0 1 3 B 7 Z y g o n e m e rt e s s p .B O B A 0 1 4 B 8 Z y g o n e m e rt e s s p .B O B A 0 1 4 B O N 8 8 Z y g o n e m e rt e s s p .B O B A 0 1 4 B O N 9 1 Z y g o n e m e r te s s p .B O B A 0 1 4 B O N 6 2 Z y g o n e m e r te s s p .B O B A 0 1 5 B O N 6 3 Z y g o n e m e r te s s p .B O B A 0

08 Figure 2 .
Figure 2. Neighbor-joining tree of COI sequences.Sequences generated in this study are shown in blue.BOBA numbers reflect subsets (OTUs) from the ASAP analysis.

1 Figure 3 .
Figure 3. Neighbor-joining tree of 16S sequences.Sequences generated in this study are shown in blue.BOBA numbers reflect subsets (OTUs) from ASAP analysis on COI data.

western
Pacific and Atlantic oceans, and the Baltic, North, and Mediterranean seas, it has been shown to constitute a large cryptic species complex with at least six lineages in Europe (Verdes et al. 2021).The seventh, NE Pacific, lineage most certainly represents an undescribed species.At least one closely related look-alike, Cerebratulus sp."spade head", occurs in southern Oregon (Hiebert 2016; Maslakova et al. 2022: OTU 63) and another, recently re-described as Cerebratulus orochi Kajihara, 2020, in the Sea of Japan.

Figure 6 .Figure 7 .
Figure 6.Hoplonemerteans of Bodega Bay A Nipponnemertes sp.BOBA028, a new species, individual BON95.Dorsal and ventral (inset) view of the anterior, showing cerebral organ furrows with numerous secondary furrows (arrowhead) and pink cerebral ganglia B Amphiporus sp.BOBA024, a new species, individual BON61.Inset a close up lateral view of the anterior, showing red blood vessels (arrowhead) C, D Amphiporus sp.BOBA018 individual BON44 D close up of head in transmitted light, showing pattern of ocelli E, F Amphiporus sp.BOBA017 individual B20 F close up of head in transmitted light, showing pattern of ocelli G Poseidonemertes sp.BOBA010, a new species, individual B14.Inset a close up of the head showing ocelli H Poseidonemertes sp.BOBA033, a new species, individual BON35, full body.Inset a close up of the head showing ocelli I Tetrastemma nigrifrons, anterior end of BON01 J Tetrastemma sp.BOBA029, close up of head of B04 in transmitted light K Zygonemertes sp.BOBA012, individual B09 L Zygonemertes sp.BOBA014, a new species, close up of anterior of BON88 in reflected light, showing post-cerebral ocelli (arrowheads) M, N Zygonemertes sp.BOBA013, individual BON87 N close up of anterior showing post cerebral ocelli (arrowheads) O Zygonemertes sp.BOBA015, a new species, individual BON63 P Antarctonemertes phyllospadicola, individual BON67 Q Nemertellina sp.BOBA011, new to science, individual BON69 R-S Oerstedia sp.BOBA022, new to science, showing differences in color pattern R individual BON32 S individual BON33 T Tetrastemma bilineatum, individual B18.

Table 1 .
Nemertean collection locations in the Bodega Bay region.Locations #8 and #11-#18 are within the Bodega Marine Reserve.

Table 2 .
Species identity, collection location, and accession numbers of nemertean specimens from Bodega Bay, CA.

Table 3 .
PCR primers used in this study.

Table 4 .
Nemertean species identified from Bodega Bay, California.The term "unresolved" refers to cryptic species of uncertain taxonomic status.
The two Poseidonemertes specimens from this study (B14 and BON35 listed below) resemble Poseidonemertes collaris Roe & Wickham, 1984 described from Bodega Bay, California, and other light-colored members of the genus, e.g., Poseidonemertes maslakovae Chernyshev, 2002 and Poseidonemertes sp.508 from the Sea of Japan.P. collaris is the only member of the genus previously reported from the Pacific coast of the U.S. COI sequence data suggest B14 represents a distinct OTU (8.2% divergent) from what was reported as P. collaris by Thollesson and Norenburg (2003) and from BON35 (described below, 19% divergent), or any other previously sequenced members of the genus.Habitat.Collected just offshore, < 200 m from an open coast beach, among subtidal sand/mud sediments from a depth of 6-7 m (station 3).Distribution.Bodega Bay, CA, USA (this study).Notes.Species new to science.ZooKeys 1204: 15-64 (2024), DOI: 10.3897/zookeys.1204.117869Christina I. Ellison et al.: Nemertean diversity of Bodega Bay Chernyshev et al. 2021aylogenetic analysis.Tetrastemma nigrifrons is part of the Tetrastemma clade sensuChernyshev et al. 2021a.The other two species are included here tentatively.