Hidden in plain sight, Chaetopterus dewysee sp. nov. (Chaetopteridae, Annelida) – A new species from Southern California

We describe a long-unnamed Chaetopterus Cuvier, 1830 species from southern California, using a combination of DNA barcoding and detailed morphological investigation employing highresolution X-ray microtomography (micro-CT). Chaetopterus dewysee sp. nov. is not only one of the most dominant annelids in the benthic communities of the shallow end of the La Jolla submarine canyon, but also a well-established model for studying bioluminescence and has a published transcriptome. The description and naming of this southern Californian Chaetopterus is a step towards the muchneeded revision of the group’s taxonomy and towards resolving the confusion over the ʻcosmopolitanʼ Chaetopterus variopedatus species complex. Micro-CT data showing details of both internal and external anatomy has been made freely available as the fi rst annelid cybertype.

Chaetopterid taxonomy is not reliably resolved and a comprehensive revision is needed (Osborn et al. 2007; Moore et al. 2017). Especially within Chaetopterus, there is repeated confusion, mainly due to the synonymization of many species into a widely distributed and highly variable species C. variopedatus (Renier, 1804) by Fauvel (1927) and Hartman (1959), which has a type locality in the Mediterranean. In many other regions around the world, the name C. variopedatus was applied and no new species were named. Such is the case for US west coast, where Treadwell (1914) saw no reason not to use the European name for specimens he studied from southern California. This has been followed ever since for Chaetopterus from this region, with numerous records (see Hartman 1969) and studies (e.g., Brown 1975Brown , 1977Sumida 1983) referring to C. variopedatus, or more recently to Chaetopterus sp. (e.g., Deheyn et al. 2013;Weigand et al. 2017).
This taxonomic ʻlumpingʼ into a large, cosmopolitan species with an implied broad dispersal capacity was justifi ed by the long planktonic stage of chaetopterid larvae in the water column (Scheltema 1974). However, it has been suggested repeatedly that C. variopedatus is a species complex of both morphologically (Petersen 1984a(Petersen , 1984bPetersen & Britayev 1997) and molecularly distinct species (Osborn et al. 2007;Martin et al. 2008). Herein, we describe a long-unnamed Chaetopterus species from southern California. Chaetopterus dewysee sp. nov. occurs in large densities and makes up a large portion of the benthic biomass in the shallow reaches of the La Jolla submarine canyon (Fig. 1D). It is one of the most abundant and visually conspicuous annelid species with a reported population density of on average 20.8 individuals per m 2 in Fisherman's Cove, Santa Catalina (Chess & Hobson 1997). In addition to being one of the most dominant annelids in benthic communities, Chaetopterus dewysee sp. nov. is established as a model in the Scripps Institution of Oceanography and has been the focus of many studies investigating the biochemistry of light production and the bioluminescent properties of Chaetopterus mucus (Deheyn et al. 2013;Shah et al. 2014Shah et al. , 2015Branchini et al. 2014;Rawat & Deheyn 2016;Weigand et al. 2017). Chaetopterus dewysee sp. nov. also has a sequenced transcriptome (Accession No: SRX755856) and was included (as Chaetopterus sp. nov.) in large scale phylogenomic studies of Annelida Lemer et al. 2015). Together with our description of C. dewysee sp. nov. we provide the fi rst three-dimensional annelid ʻcybertypeʼ. This micro-computed tomography dataset is freely available for future taxonomic and morphological investigations.

Morphology
Live specimens were studied and photographed with a Leica MZ9.5 stereo microscope mounted with a Canon EOS Rebel T5i digital camera. Chaetigers were dissected and placed on separate microscope slides, 50% bleach was used to dissolve the tissue. The tissue was slightly teased away from the chaetae. These were examined and photographed with a Leica DMR HC compound microscope. All animals were relaxed using MgCl 2 , fi xed with 10% formaldehyde in seawater for a few days, rinsed in fresh water and transferred to 70% alcohol. Posterior parapodia were subsampled for DNA and fi xed directly in 95% ethanol.

Micro-CT
The specimen fi xed for micro-CT was preserved in 50% ethanol. In order to stain soft tissue and increase contrast, the specimen was transferred to a 0.3% phosphotungstic acid (PTA) solution in 70% ethanol (Metscher 2009). The specimen remained in this solution for 3 months and was scanned using a Skyscan TILIC E. & ROUSE G.W., Chaetopterus dewysee sp. nov.
3 1272 (Bruker microCT, Kontich, Belgium) with the following scan parameters: 60 kV source voltage, 166 μA source current, 741 ms exposure and a camera resolution of 1632 × 1092 px. The voxel resolution was 8 μm. An aligned image stack was generated with the software Nrecon (Bruker) and the surface renderings were generated with the software Drishti 2.6.5. (National University, Canberra, Australia). Micro-CT data together with a 3D surface rendering are deposited online in the morphological data repository MorphDBase (Grobe & Vogt 2009).

DNA sequences
DNA was extracted from posterior parapodia of animals using the Zymo Research Quick-DNA™ Miniprep kit. DNA from the larva of Chaetopterus dewysee sp. nov. was extracted using the Quick-DNA™ Microprep kit. Up to 686bp of mitochondrial cytochrome c oxidase subunit I (COI) were amplifi ed using the polyLCO/polyHCO primer set (Carr et al. 2011). Amplifi cation was carried out using 8.5 μl of ddH2O, 12.5 μl of ApexTM 2.0x Taq RED DNA Polymerase Master Mix (Genesee Scientifi c), 1 μl each of the forward and reverse primers (10 μM), and 2 μl of eluted DNA. The reactions were carried out in an Eppendorf thermal cycler. The COI reaction protocol was as follows: 94°C / 60 s -(94°C / 40 s -45°C / 40 s -72°C / 60 s) * 5 cycles -(94°C / 40 s -51°C / 40 s -72°C / 60 s) * 35 cycles -72°C / 300 s. Successfully amplifi ed products were purifi ed using 2 μl of ExoSAP-IT PCR product cleaning reagent. The cleaned products were then sequenced by Eurofi ns Genomics (Louisville, KY) and assembled with Geneious ver. 11.0.2 (https://www.geneious.com). The COI sequence was also pulled from the assembly of the published Chaetopterus transcriptome (SRX755856) using the direct sequencing results as a blast query. Other available Chaetopterus COI sequences were acquired from GenBank, mostly from the Moore et al. (2017) study, but also including one C. dewysee sp. nov. sequence as, "Chaetopterus sp. 1", from Osborn et al. (2007) collected from Santa Barbara, California. Sequences of the sister group, Mesochaetopterus Potts, 1914, were used to root the phylogenetic analysis, following Moore et al. (2017).

Diagnosis
Chaetopterus dewysee sp. nov. is characterized by having a long u-shaped tube partly buried in sediment, 10 region A chaetigers, 11-12 club-shaped a4 cutting chaetae with dark brown, coppery metallic coloration, a patch of notopodial uncini at the upper ventral margin of the modifi ed b3-b5 notopodia.

Etymology
Named for Mary 'Dewy' White, for her support of the Rouse lab and her passion for conservation and marine biological research. Based on her love of the sea we have incorporated the German word 'See' into the name.

Tube
Parchment like, U-shaped tube, sometimes with sand debris and shell fragments on the outer surface (Fig. 1B). Both tube openings almost half in diameter (8 mm) compared to the middle section of the tube (16 mm). Total tube length 293 mm.

Habitat
Commonly found partially buried along canyon walls in large assemblages of solitary, intermingled tubes and sediment with other fauna, such as sponges and tunicates.     nov. and C. variopedatus (Renier, 1804) from the type locality are in bold. Number of sequences included for each terminal is in brackets. Details on sequences that were analyzed can be found in Table 1. Haplotype network for the nine Chaetopterus dewysee sp. nov. sequences is shown next to the tree.

Molecular information
All type specimens of Chaetopterus dewysee sp. nov., except for the cybertype, were subsampled and sequenced for COI (Table 1). The specimen chosen for the micro-CT scan was kept intact as a cybertype. All COI sequences for specimens identifi ed as Chaetopterus dewysee sp. nov. were > 98.8% similar. These included the COI sequence pulled from the available Chaetopterus transcriptome, the sequence from Santa Barbara, published by Osborn et al. (2007) and the COI sequence from the larva (Fig. 1C). The haplotype network for the nine C. dewysee sp. nov. sequences (Fig. 5) shows minor variation amongst the specimens. The most frequent haplotype is shared by four individuals (the sequenced larva, the holotype A11476, A10193 and A11653). In addition to this, there are 5 low-frequency haplotypes, each represented by a single specimen. The haplotypes are separated by one to three mutational steps.
On the maximum likelihood tree sequences that were more than 97% similar were given the same terminal name and the branches were collapsed (Fig. 5). Average identity between the COI sequences of diff erent Chaetopterus spp. was 78.7% (min. 72.1%, max 85.2%).

Remarks
Chaetopterus dewysee sp. nov. most resembles the two European species, C. brevis Lespés, 1872 and C. variopedatus. The main diff erence between the 3 species is in the morphology of a4 cutting chaetae. Chaetopterus variopedatus has a4 cutting chaetae with teeth, whereas the cutting chaetae of C. brevis are symmetrical and distally infl ated. In C. dewysee sp. nov. the cutting chaetae have a smooth, asymmetrical tip with a sharp apical point. Furthermore, region C notopodia of C. dewysee sp. nov. have no lateral cirrus, which is present in both C. brevis and C. variopedatus. Chaetopterus brevis also diff ers from the other two species in having a gregarious habit, that can be occupied by multiple individuals. Relationships were not supported across most of the phylogeny generated here using COI (Fig. 5).
Chaetopterus dewysee sp. nov. is sister group to Chaetopterus cf. brevis from France, but with low support. In Moore et al. (2017), which also used nuclear 18S and 28S data, this relationship was also recovered, but with very strong support.

Discussion
The concept of ʻcybertypesʼ was introduced by Faulwetter et al. (2013) and in the same paper the potential of micro computed tomography as a taxonomic resource was illustrated using examples from diff erent annelid species. Micro-CT scanning has been used for species descriptions in arthropods and there are several cybertypes available for myriapods (Stoev et al. 2013;Akkari et al. 2015) and ants (Hita Garcia et al. 2017). According to the defi nition of Faulwetter et al. (2013), a cybertype needs to: (a) provide morphological and anatomical information of at least the same accuracy as a physical type, that is not linked to a specifi c research question; (b) a cybertype should be associated with an original type; (c) a cybertype has to be made freely accessible.
Virtual dissections together with contrast enhancing stains signifi cantly improve the resolution of anatomical details of otherwise inconspicuous soft-tissue (Fig. 4C, E) and micro-CT is becoming more common as a tool to study the internal anatomy of annelids (Paterson et al. 2014). The advantages of a non-invasive technique like micro-CT are clear as this technique allows a detailed imaging of historical and valuable museum specimens. Furthermore, these methods are signifi cantly less labor-intensive than traditional histology and allow a more automated workfl ow that can generate large amounts of morphological data in a shorter period of time. Another great advantage is how the volume data generated shows anatomical structures in their original arrangement and thus enables automated image processing for anatomical 3D reconstructions. Micro-CT might not be able to provide the high cell-level resolution of serial histology, but certain internal details of the myoanatomy (Fig. 4C, E), digestive system (Parapar et al. 2017), nervous system (Beckers et al. 2019) and hard structures, like jaws, in annelids (Watson & Faulwetter 2017) can be visualized in great detail.
The micro-CT dataset and 3D renderings of Chaetopterus dewysee sp. nov. (SIO-BIC A12034) we provide here constitute the fi rst annelid cybertype which is freely available for future research on this species.