A new species of the Marphysa sanguinea complex from French waters (Bay of Biscay, NE Atlantic) (Annelida, Eunicidae)

Abstract A new species of Eunicidae, Marphysa victori sp. n., has been identified from Arcachon Bay, Bay of Biscay, NE Atlantic. This new species, belonging to the sanguinea complex, is characterised by branchiae with long filaments from chaetigers 26–34, the presence of four types of pectinate chaetae with first ones present from chaetiger 2, a large number of both pectinate chaetae and compound spinigers, and the pygidium with only one pair of pygidial cirri. An identification key for European species of the genus Marphysa is provided.


Introduction
In Arcachon Bay, blood worms of the genus Marphysa Quatrefages, 1866 are widely collected as bait both by recreational and professional fishermen. Since 2011, 13 companies (with a total of 26 employees) were operating in the lagoon and they recorded that 1.3-2.5 tons/year (wet weight) of Marphysa were collected which represents approximatively 400,000 worms. In reality, around 1 million of these worms could be fished each year in the bay. Most of these worms are shipped alive by air (in boxes with plant litter) to sellers situated on the western French Mediterranean coasts. Then, they are sold to recreational fishermen and used locally. Until now, these blood worms were misidentified as Marphysa sanguinea (Montagu, 1813), which was originally described from the south coast of Devon, UK (Hutchings and Karageorgopoulos 2003).
According to Fauchald (1970), species of the genus Marphysa can be grouped into four artificial groups based on the type of compound chaetae: no compound chaetae present (Group A), only compound spinigers present (Group B), only compound falcigers present (Group C) and both compound spinigers and falcigers present (Group D). Glasby and Hutchings (2010) added a fifth group, having compound spinigers only anteriorly and posterior segments only with simple limbate chaetae, and also encapsulating embryos in jelly cocoons. Each group can also then be divided into species having branchiae present only on anterior parapodia (subdivision 1) or branchiae present over most of the body (subdivision 2). In European waters, M. sanguinea is the only representative of the Group B2 currently reported.
Marphysa sanguinea has been recorded in Arcachon Bay by numerous workers (Lafont 1871;Boisseau 1962;Auby 1991;Blanchet 2004;Salvo 2010). However, all these papers just list the fauna present as a result of ecological studies without commenting on the species. It appears that no material was deposited in a museum and so could not be examined for this study. These records are not surprising as this species is originally described from the northern coast of the English Channel. Fauvel (1923) in his monograph of French polychaetes lists M. sanguinea and provides description and illustrations but without commenting on what material he examined. Nevertheless, recent re-descriptions of this species (Hutchings and Karageorgopoulos 2003;Hutchings et al. 2012) suggest that records of this species from outside the type locality should be checked, and that many records have been misidentified and represent new species (Zanol et al. 2016(Zanol et al. , 2017. Hutchings and Kupriyanova (2017) encourage taxonomists to check carefully their specimens.
Both morphological and molecular analyses confirm the existence of an undescribed species of Marphysa in Arcachon Bay. The present paper provides the description of this species as well as a key for European described species of this genus.

Sampling and morphological analyses
Specimens examined in this study were collected in Arcachon Bay (Fig. 1) in September 2016 by hand, using a shovel and spade from intertidal mud flats. Live specimens were anaesthetized with 7% magnesium chloride (MgCl 2 ). A small piece of body was removed from several specimens and fixed in 96% ethanol for molecular studies. The rest of each specimen was fixed in 4% formaldehyde seawater solution, then transferred to 70% ethanol for morphological analyses. Preserved specimens were examined under a Nikon SMZ25 stereomicroscope and a Nikon Eclipse E400 microscope, and photographed with a Nikon DS-Ri 2 camera. Measurements were made with the NIS-Elements Analysis software. Drawings were made from pictures using Inkscape software and Wacom Intuos 5 tablet.
Selected parapodia along the body were removed from the holotype AM W.49047, dehydrated in ethanol, critical point dried, covered with 20 nm of gold, examined under the scanning electron microscope (JEOL JSM 6480LA) and imaged with a secondary detector at Macquarie University, Sydney, Australia. These parapodia included chaetiger 3 and then chaetigers at 20% intervals along the body (chaetigers 83, 163, 243, 323, 403, and 470). Additionally, the following parapodia were removed from holotype (chaetigers 4, 93, 166, 259, 352, and 445) mounted and examined under the light microscope, with permanent slides made.
The studied material is deposited at the Australian Museum, Sydney (AM) and the Muséum National d'Histoire Naturelle, Paris (MNHN). Additional material is lodged in the collection housed at the Arcachon Marine Station.

Molecular data and analyses
Sub-samples for DNA analysis were removed from live specimens, placed in ethanol 96% and frozen at -20°C. Extraction of DNA was done with QIAamp DNA Micro Kit (QIAGEN) following protocol supplied by the manufacturers. Approximately 400 bp of 16S and 700 bp of COI (cytochrome c oxidase subunit I) genes were amplified using primers diop16SF (TGCAAAGGTAGCATAATCATTTG) and diop16SR (ACTCAGATCACGTAGGA) for 16S were designed, and polyLCO and polyHCO for COI (Carr et al. 2011).
Overlapping sequence (forward and reverse) fragments were merged into consensus sequences and aligned using Clustal Omega. For COI, sequences were translated into amino acid alignment and checked for stop codons to avoid pseudogenes. The minimum length coverage was around 430bp for 16S and 660 bp for COI. All sequences obtained in this study have been deposited in GenBank (Table 1).
Pair-wise Kimura 2-parameter (K2P) genetic distance and maximum likelihood tree using K2P model and non-parametric bootstrap branch support (1000 replicates) were performed using MEGA version 7.0.26. Tree-based analysis was obtained with all Marphysa species having COI sequences available in GenBank and considering other genera of Eunicidae as outgroup (Table 1). Description (based on holotype and paratypes). Live specimens iridescent, dark red with lighter spots, prostomium appendages and parapodia green olive, end of prostomial appendages whitish, branchial filaments red and iridescent. Recently fixed specimens olive-green to brown with lighter spots, prostomium appendages and parapodia pinkish. Preserved holotype with brown mottled pigmentation anteriorly increases in intensity towards prostomium, antennae, and palps whitish. Body long, with same width throughout, slightly tapering at anterior and posterior ends. Prostomium shorter than anterior ring of peristomium, as wide as peristomium, bilobed with buccal lips separated by deep ventral and dorsal notch with each lobe rounded with base of them strongly pigmented ( Fig. 2A-C). Anterior ring of peristomium longer than posterior ring (2.2 to 3 times) ( Fig. 2A, C). Eyes present, positioned between palps and lateral antennae (Fig. 2B), faded in larger specimens (not visible on holotype). Prostomial appendages smooth, arranged in horseshoe, slightly tapering; median antenna longer than lateral antennae, palps shortest appendages (paratypes exhibit considerable variation in the ratio of length of median and lateral antennae and of palps to a lesser extent). Antennal styles and palpostyles smooth although surface slightly wrinkled. MxI more than twice as long as carrier and five times longer than closing system. MxIII at least in part located ventral to MxII. Attachment lamella of MxIII short with irregular shape, placed at the middle of the plate. Left MxIV with attachment lamella semicircular, situated along posterior edge. Right MIV with attachment lamella semicircular, more developed in the central portion, situated along posterior edge. Maxillary formula: I=1+1, II=5+5, III=5-6+6-7, IV=3-4+0, V=1+1 (Fig. 3E).
Pygidium with only one pair of long pygidial cirri on ventral margin (approximately as long as last 15 segments), anus slightly crenulated with 12 small indentations (Fig. 2D).
Morphological variations. Paratypes with branchiae starting from chaetigers 26 (MNHN-IA-TYPE 1808) to 28 for smaller specimens and from 28 to 34 for larger ones. Eyes clearly visible on small (MNHN-IA-TYPE 1807 and MNHN-IA-TYPE 1808) and medium specimens, more difficult to see on larger ones. One exceptional specimen (MNHN-IA-TYPE 1805) with one pair of small papillae in addition to one pair of pygidial cirri, with papillae placed more ventrally than cirri.
Etymology. This species is named after Victor Lavesque, first and second authors' son. Type locality. NE Atlantic, France, Arcachon Bay.
Habitat. Intertidal on mudflats, under or close to oyster reefs or abandoned oyster farms, 5 to 60 cm depth. Few specimens were found in galleries into old piece of driftwood.  (Table 1).
As the identification of Marphysa species from the sanguinea group is very complex, molecular tools are very important. First of all, comparison of COI and 16S sequences confirmed that M. victori sp. n. was different from M. sanguinea (census Zanol et al. 2014) (Fig. 5): interspecific pairwise genetic distances were 21.8% for COI and 15.2% for 16S. Secondly, molecular analysis clearly distinguished M. victori sp. n. from other species with sequences available in GenBank (Fig. 5). Finally, they permitted us to link morphological differences to age of specimens. Indeed, intraspecific pairwise genetic distance was zero among specimens and allow to conclude that intensity of eye pigments and the segment on which the branchiae first appear was related to the size (age) of worms and not to the presence of different species.

Discussion
Marphysa sanguinea (Montagu, 1813) is the type species of the genus and has been widely reported from around the world. This is partly because the original description was very brief and poorly illustrated, and because all species superficially look similar. Hartman (1959) complicated the issue by synonymising several species with M. sanguinea with no explanation and the species joined a list of so-called "cosmopolitan species" (Hutchings and Kupriyanova 2017). Hutchings and Karageorgopoulos (2003) while trying to resolve the true identity of a commercially important species in Moreton Bay (Queensland, Australia) which had always been called M. sanguinea, examined material from SW England and designated a neotype of M. sanguinea and then redescribed the species. This then allowed the species from Moreton Bay to be described as a new species, and subsequently other species of Marphysa were described from along the east coast of Australia (Zanol et al. 2016(Zanol et al. , 2017. In the same way, a new species of the sanguinea group from French Atlantic coasts is described in this study. In Europe, only two species belong to the B2 group (Fauchald 1970). Marphysa victori sp. n. can be distinguished from M. sanguinea in having branchiae from chaetiger 26-34 (instead of 13-27), pectinate chaetae of four types (instead of two types, with an absence of anodont, asymmetrical pectinate chaetae with 2-4 large teeth), lacking subacicular hooks (see key above). Moreover, the neotype of M. sanguinea was described from south coast of England living intertidally in rocks which are easily identified and which are in a very different habitat to that of M. victori sp. n. (mudflats).
Three species are known to occur in Arcachon bay: M. bellii, M. fallax, and M. sanguinea. In the absence of specimens stored in a collection, it is very difficult to know how long M. victori sp. n. has been present in Arcachon Bay and confused with M. sanguinea. Moreover, the hypothesis that this new species is a non-indigenous species (NIS) cannot be completely dismissed. Arcachon Bay is one of the major French oyster farming sites with a production of 7,000-8,000 t per year of exotic Pacific cupped oyster Crassostrea gigas (Thunberg, 1793). In the early 1970s, the Portuguese cupped oyster Crassostrea angulata (Lamarck, 1819), which has been farmed in the bay since the end of the 19 th century, was decimated by a viral disease (Goulletquer et al. 2002). To sustain the local oyster industry, the exotic Pacific cupped oyster C. gigas was then introduced into Arcachon Bay between 1971 and 1975, as spat from Senday Bay, NE Honshu Island, Japan (1,176 t of spat collectors from 1971 to 1975) and as adults from British Columbia, Canada (137.5 t from 1971 to 1973) (Grizel and Héral 1991). Several non-indigenous species, probably introduced with oyster transfers, were recently found in Arcachon Bay (see references in Gouillieux et al. 2016). As M. victori sp. n. is very abundant close to oyster reefs, specimens could have been hitchhiked in oyster shells coming from Japan or Pacific coasts of USA. Worldwide, among the B2 group, only two species are characterized by the presence of four types of pectinate chaetae: Marphysa multipectinata (Liu, Hutchings & Sun, 2017) recently described from south coast of China and M. victori sp. n. Marphysa multipectinata differs from M. victori sp. n. in the appearance of pectinate chaetae (from chaetiger ~ 70 instead of chaetiger 2 for M. victori sp. n.), the maximal number of spiniger compound chaetae (27 vs >40 for M. victori sp. n.) and pectinate chaetae (22 vs 34 for M. victori sp. n.), the presence of subacicular hooks, the absence of inflated base of ventral cirri, the number of teeth of Mx II (3+3 vs 5+5 for M. victori sp. n.) and the presence of two pairs of pygidial cirri (instead of one single pair for M. victori sp. n.).
Alternatively, M. victori sp. n. could be native from Arcachon Bay and subsequently have been introduced into other European localities. Indeed, local oyster farmers often transfer their spat and juveniles between rearing areas in France (both on the Atlantic and Mediterranean coasts) (Goulletquer et al. 2002) or even to other European countries (Gouillieux et al. 2016). Moreover, thousands (probably millions) of specimens of M. victori sp. n. are shipped alive with litter to resellers situated on the western French Mediterranean coasts and then are used as bait by anglers. Recent studies have highlighted the possibility of these practises facilitating the introduction of invasive species (Olive 1994). Bait worm packaging are considered to be an important vector for transporting non-native algae, microorganisms and other invertebrates (Haska et al. 2012;Fowler et al. 2016). For example, a total of 114 taxa have been identified by Fowler et al. (2016) in baitworm shipments from Maine. Moreover, use of live baits contributes to the dispersal of worms in new marine ecosystems. Kilian et al. (2012) showed that a certain number of anglers in Maryland (USA) released their unused baits into the water at the end of a fishing trip. In this way, M. victori sp. n. might become a non-indigenous species in the Mediterranean Sea. Finally, the presence of M. victori sp. n. in driftwood could also lead to an extension of its geographical distribution in the Bay of Biscay via water currents.
To conclude, we suggest that all records of Marphysa from northern Europe need to be carefully checked to see if they represent a currently known species including M. sanguinea or represent an undescribed species. As well, the pattern of chaetal arrangement along the body need to be examined under the SEM, and combined with molecular data to correctly identify these species which often superficially resemble each other and vouchers need to be deposited in a museum.